KR102076777B1 - A composition comprising a Gas6 protein or an receptor activator thereof for preventing or treating fibrosis - Google Patents

Abstract

The present invention relates to a composition for the prevention or treatment of fibrosis comprising a Gas6 protein or a receptor activator thereof.
Gas6 proteins induce epithelial production of PGE ₂ , PGD ₂ and HGF through their receptors in epithelial cells, inhibit EMT in a self-secreting manner via their downstream signaling pathways, thus preventing and treating fibrosis Therefore, Gas6 protein and its receptor activator have a very good effect for the prevention or treatment of fibrosis.

Description

Composition or composition for preventing or treating fibrosis comprising gas6 protein or an receptor activator thereof

The present invention relates to a composition for the prevention or treatment of fibrosis comprising a Gas6 protein or a receptor activator thereof.

Fibrosis is a disease in which abnormal production, accumulation, and deposition of extracellular matrix by fibroblasts occurs, and is caused by fibrosis of organs or tissues. Fibrosis is a very fatal disease that causes organ damage. Idiopathic pulmonary fibrosis (IPF), for example, is the result of recurrent alveolar epithelial cell damage associated with fibroblast accumulation and myofibroblast differentiation, and is accompanied by an extracellular matrix (ECM) with irreversible destruction of lung parenchyma tissue. ) Is a chronic, progressive, and lethal disease that causes overaccumulation. However, to date, there is no effective treatment for fibrosis (Fibrogenesis Tissue Repair, 2012, 5 (1): 11; N Engl J Med, 2001, 345 (7): 517), which is a therapeutic agent that can effectively prevent or treat fibrosis. Development is constantly required.

As a result of our diligent efforts to develop a composition for the prevention or treatment of fibrosis, the present inventors have studied the new role of Gas6 protein and the mechanism of action of Gas6 protein on the epithelial to mesenchymal transition (EMT), migration and invasion of alveolar type 2 epithelial cells. The present invention was completed by identifying and confirming the prophylactic and therapeutic effects of Gas6 in a bleomycin-induced lung fibrosis mouse model.

One object of the present invention is to provide a composition for the prevention or treatment of fibrosis, comprising a Gas6 protein or a receptor activator of Gas6 protein.

Another object of the present invention is to provide a method for preventing or treating fibrosis, comprising administering a Gas6 protein or a receptor activator of Gas6 protein to a subject.

This will be described in detail below. Meanwhile, each of the descriptions and the embodiments disclosed in the present invention may be applied to each of the other descriptions and the embodiments. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. In addition, the scope of the present invention is not to be limited by the specific description described below.

One aspect of the present invention for achieving the above object is a pharmaceutical composition for the prevention or treatment of fibrosis, comprising as an active ingredient Gas6 (Growth arrest-specific 6) protein or Gas6 protein receptor activator.

As used herein, the term "fibrosis" means that excess fibrous connective tissue is formed in an organ or tissue. It can be distinguished from fibrous tissue as a normal component in organs or tissues. Due to the excessive accumulation of extracellular metrix such as fibronectin, collagen, etc. by fibroblasts, fibrosis can be understood as a fatal disease that eventually causes organ damage.

As used herein, the term "epithelial to mesenchymal transition (EMT)" refers to a phenomenon in which epithelial cells are converted into mesenchymal cells and is associated with embryonic development, organ development, wound healing and stem cell behavior, and pathology in organ fibrosis and cancer development. Is known to contribute to human health (J Clin Invest, 2009, 119 (6): 1420). Fibroblasts are characterized by (1) proliferation and differentiation of resident lung fibroblasts, (2) EMTs that convert from AEC (alveolar epithelial cells) to (muscle) fibroblasts, and (3) differentiation of myeloid progenitor cells, Similarly, it has been reported to have a variety of origins (Curr Rheumatol Rep, 2006, 8: 145), and the results reported to date suggest that EMT is a major phenomenon in the development of fibrosis such as IPF (Am J Pathol, 2009, 175: 3; J Exp Med, 2011, 208: 1339. Therefore, it can be seen to be obvious to those skilled in the art that EMT inhibition may have a prophylactic and therapeutic effect on fibrosis.

In the present invention, the fibrosis is lung, kidney, liver, heart, brain, blood vessels, joints, intestine, skin, soft tissue, bone marrow, penis, peritoneum, lens, muscle, spine, testes, ovaries, breast, thyroid, tympanic membrane, pancreas, Fibrosis-related disorders that can appear in all tissues, such as the gallbladder, bladder, or prostate Specifically, in the present invention, fibrosis is a disease caused by fibrosis occurring in each tissue of the body, such as abnormal wound healing, alcoholic liver injury-induced liver fibrosis, connective fibrosis, Crohn's disease (intestinal fibrosis), pancreatic and lung cystic Fibrosis, especially injection fibrosis, endocardial myocardial fibrosis or cardiac fibrosis, fibrosis caused by Graft-Versus-Host Disease (GVHD), fibrosis of the spleen, retinal fibrosis, which can occur as a complication of intramuscular injection in children. Fibrosis of the eyes, including fibrosis complications of surgery or injection fibrosis, glomerulonephritis, interstitial fibrosis, keloids and hypertrophic scars (skin fibrosis), macular degeneration, mediastinal fibrosis (soft tissue fibrosis of the mediastinum), focal sclerosis (morphea), multifocal Fibrosis, myeloid fibrosis, renal systemic fibrosis (skin fibrosis), nodular epidermal fibrosis (eg, benign fibrous histiocytes) , Pleural fibrosis, fibrosis, proliferative fibrosis, pipestem fibrosis, posterior fibrosis, progressive multiple fibrosis (complications of pulmonary fibrosis, coal worker pneumoconiosis) as a result of surgery (e.g. surgical implantation), Old myocardial infarction (heart fibrosis), pancreatic fibrosis, progressive multiple fibrosis, radiation fibrosis, renal fibrosis, renal fibrosis associated with or causing chronic kidney disease, retroperitoneal fibrosis (fibrosis of the soft tissue of the peritoneum), postoperative scarring, Scleroderma / systemic sclerosis (skin fibrosis), epithelial cellosis, uterine fibrosis, or viral hepatitis-induced fibrosis.

In the present invention, pulmonary fibrosis is related to idiopathic Pulmonary Fibrosis, Nonspecific Interstitial Pneumonia, Acute Interstitial Pneumonia, Cryptogenic Organizing Pneumonia, Respiratory bronchiolitis Respiratory Bronchiolitisassociated Interstitial Lung, Desquamative Interstitial Pneumonia, Lymphoid Interstitial Pneumonia, Interstitial Pulmonary Fibrosis, and Diffuse Pulmonary Fibrosis It is not.

As used herein, the term "treatment" means any action that improves or beneficially alters the symptoms of fibrosis by administration of the pharmaceutical composition, and "prevention" inhibits the onset of fibrosis by administration of the pharmaceutical composition. Or any action that delays or delays.

In the present invention, it was confirmed for the first time that the activator of the Gas6 protein or Gas6 protein receptor can suppress the EMT phenomenon, it was found that the activator of the Gas6 protein or Gas6 protein receptor can be used for the prevention and treatment of fibrosis.

As used herein, the term "Gas6 (Growth arrest-specific protein 6)" refers to a secretory vitamin K-dependent protein, also named as AXSF or AXLLG. Gas6 protein in the present invention, for example, may have an amino acid sequence (SEQ ID NO: 35) of the NCBI accession number (NP_062394.2), but is not limited thereto, and has a homology with a known Gas6 protein, and is confirmed in the present invention Some sequences may be deleted, added, substituted, or Gas6 proteins derived from humans or other animals as long as they have the same activity, and all of them may be included within the scope of the present invention. In the present invention, sequence homology may be identical to at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% of the wild type Gas6 protein. In addition, fragments obtained by cleaving some domains for activity in the wild-type Gas6 protein, and fusion protein forms combining known peptides to increase the expression, isolation, purification, or activity of the protein, have the same activity as the wild-type Gas6 protein. One may fall within the scope of the Gas6 protein of the invention. Gas6 protein in the present invention may be prepared through methods known in the art, for example, peptide synthesis or production of proteins using transformed cells, but is not limited thereto. In the examples of the present invention, a mouse Gas6 protein of SEQ ID NO: 35 was synthesized and used (R & D Systems; Minneapolis, MN, USA).

Gas6 contains an N-terminal gamma-carboxyglutamic acid (Gla) domain, four EGF (epidermal growth factor) -like domains, and a large C-terminal region homologous to sex hormone binding globulin (SHBG). Biol, 1993, 13: 4976; Blood Cells Mol Dis, 2006, 36: 373). Gas6 is expressed in lung, heart, kidney, intestine, fibroblasts, endothelial cells, bone marrow cells, vascular smooth muscle, leukocytes, and neurons, and is known as a common ligand of the TAM (Tyro3 / Axl / Mer) receptor subfamily (Cell, 1995, 82: 355; Nature Reviews Immunology, 2008, 8: 327). Thus, Gas6 protein receptors in the present invention include, but are not limited to, AXL receptor tyrosine kinase, Mer tyrosine kinase or TYRO3. These receptors all share significant domain similarity, including two extracellular N-terminal immunoglobulin-like domains and two fibronectin-III-like domains followed by a tyrosine kinase domain located at the end of the receptor C-terminal cytoplasm . TAM activity by Gas6 has been reported to induce signals mediating cell survival, proliferation, phagocytosis, differentiation, platelet function and thrombus stabilization. Recent studies have also shown that Gas6 signaling in macrophages and dendritic cells modulates immune responses by regulating TLR-induced cytokine secretion. Furthermore, the present inventors have recently found that epithelial cell proliferation and wound repair are performed through the paracrine role of HGF induced by the Mer / RhoA / Akt / MAP kinase signaling pathway in macrophages by reprogramming with Gas6. Although it can be promoted, the direct role of Gas6 on epithelial cell homeostasis is still unknown.

In the present invention, the activator of the Gas6 protein receptor refers to a substance capable of activating the Gas6 protein receptor to activate its downstream signaling pathway. In particular, for the purposes of the present invention, PEG2, PGD ₂ or HGF may be produced and secreted. Refers to an activating substance for a signaling pathway For example, the activator of the Gas6 protein receptor may be Protein S (Pros1), Tubby and tubby-like protein 1, or Galectin3, but is not limited thereto.

In a specific embodiment of the present invention, the effect of Gas6 inhibiting TGF-β-induced EMT in alveolar and renal epithelial cells was directly confirmed through changes in the cell morphology and mRNA and protein levels of EMT markers (FIG. 1 to 1). 4). In another embodiment, the anti-EMT activity of Gas6 was confirmed by blocking Smad independent TGF-β signal including ERK and Akt pathways (FIGS. 5 to 10). In addition, it was confirmed that Gas6 treatment induces PGE ₂ , PGD ₂ and HGF secretion, and exhibits anti-EMT effects mainly through EP2, DP2 and c-Met receptors (FIGS. 11-53). Gas6 interacted with Axl or Mer receptor tyrosine kinase in the cell membrane to block TGF-β signal through COX-2-derived PGE ₂ , PGD ₂ and RhoA-dependent HGF. (FIGS. 5-62). In particular, even in mouse primary alveolar epithelial cells, as shown in mouse alveolar epithelial cells, the anti-EMT effect of Gas6 dependent on PGE ₂ , PGD ₂ and HGF signal was confirmed (FIGS. 63 to 75). Furthermore, similar anti-EMT effects were observed upon administration of human Gas6 recombinant protein in human lung adenomas (A549 cells) (FIGS. 76-78). In another embodiment, it was confirmed that pulmonary fibrosis was improved by Gas6 administration in an animal model in which pulmonary fibrosis was induced (FIGS. 79 to 88).

Therefore, the composition of the present invention can inhibit EMT (epithelial to mesenchymal transition) by producing and secreting PEG2, PGD ₂ or HGF in the cell in a self-secreting manner, and has an excellent effect on the prevention and treatment of fibrosis, It can be very useful for prophylactic or therapeutic purposes.

In addition, the pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients or diluents commonly used in the manufacture of pharmaceutical compositions. Compositions comprising a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. When formulated, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used. Solid form preparations for oral administration may include tablet pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or lactose in one or more compounds. It can be prepared by mixing lactose, gelatin and the like. In addition to the simple excipients, lubricants such as magnesium stearate, talc and the like can also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin, may be used. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

In addition, the pharmaceutical composition of the present invention is not limited thereto, but tablets, pills, powders, granules, capsules, suspensions, solvents, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized It may have any of the formulations selected from the group consisting of formulations and suppositories.

The composition may be administered at a dose of 100 μg / kg to 500 μg / kg, but is not limited thereto. The composition of the present invention may be administered in combination with other known fibrosis treatment agents at different times or simultaneously, or the known methods of treating fibrosis may be applied together. The composition may also be administered single or multiple. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with a minimum amount without side effects, which can be readily determined by one skilled in the art.

As used herein, the term "administration" refers to introducing a pharmaceutical composition of the present invention to a subject by any suitable method, and the route of administration may be administered via various routes, oral or parenteral, as long as the target tissue can be reached. .

The pharmaceutical composition may be appropriately administered to the subject according to conventional methods, routes of administration, and dosages used in the art, depending on the purpose or need. Examples of routes of administration may be administered orally, parenterally, subcutaneously, intraperitoneally, intrapulmonally, and intranasally, and parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In addition, an appropriate dosage and frequency of administration may be selected according to methods known in the art, and the amount and frequency of administration of the pharmaceutical composition of the present invention to be administered may be based on the type of symptom to be treated, route of administration, sex, and health condition. , Diet, the age and weight of the individual, and the severity of the disease may be appropriately determined.

As used herein, the term "pharmaceutically effective amount" means an amount sufficient to inhibit or mitigate the increase in vascular permeability at a reasonable benefit / risk ratio applicable to medical use, and the effective dose level may be determined by subject type and severity, age, It may be determined by sex, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrently used drugs, and other factors well known in the medical arts.

As used herein, the term "individual" means any animal, including humans, that possesses or develops a fibrotic disease of the present invention. By administering a pharmaceutical composition of the present invention to a subject, fibrosis can be prevented or treated.

Another aspect of the invention is a method of preventing or treating fibrosis, comprising administering a Gas6 protein or a receptor activator of Gas6 protein to a subject.

The use of Gas6 protein, a receptor activator thereof, for the prevention or treatment of fibrosis is as described above.

Gas6 proteins induce epithelial production of PGE ₂ , PGD ₂ and HGF through their receptors in epithelial cells, inhibit EMT in a self-secreting manner via their downstream signaling pathways, thus preventing and treating fibrosis Therefore, Gas6 protein and its receptor activator have a very good effect for the prevention or treatment of fibrosis.

Figure 1 shows the results of confirming the shape of LA-4 cells by TGF-β and Gas6 treatment by phase contrast microscope. After treatment with 400 ng / ml Gas6 for 20 hours, 10 ng / ml of TGF-β was treated for 48 hours or 72 hours.
Figure 2 shows the results of confirming the expression level of EMT marker protein in LA-4 cells following TGF-β and Gas6 treatment. After treatment with 400 ng / ml Gas6 for 20 hours, 10 ng / ml of TGF-β was treated for 48 hours or 72 hours. * P <0.05 compared to control; + P <0.05, compare displayed values.
Figure 3 shows the results of confirming the expression level of EMT marker mRNA in LA-4 cells following TGF-β and Gas6 treatment. After treatment with 400 ng / ml Gas6 for 20 hours, 10 ng / ml of TGF-β was treated for 48 hours or 72 hours. * P <0.05 compared to control; + P <0.05, compare displayed values.
Figure 4 shows the results of confirming the expression level of EMT marker protein in HEK-293 renal epithelial cells following TGF-β and Gas6 treatment. After treatment with 400 ng / ml Gas6 for 20 hours, 10 ng / ml of TGF-β was treated for 72 hours. * P <0.05 compared to control; + P <0.05, compare displayed values.
5 shows the results of confirming the expression levels of Snai1 / 2, Zeb1 / 2 and Twist1 mRNA in LA-4 cells following TGF-β and Gas6 treatment. After treatment with 400 ng / ml Gas6 for 20 hours, 10 ng / ml of TGF-β was treated for 48 hours or 72 hours.
6 shows the results of confirming Snai1 / 2, Zeb1 / 2, and Twist1 mRNA expression levels in HEK-293 cells following TGF-β and Gas6 treatment. After treatment with 400 ng / ml Gas6 for 20 hours, 10 ng / ml of TGF-β was treated for 72 hours.
Figure 7 shows the results confirming the phosphorylation of Smad2 or Smad3 protein in LA-4 cells following TGF-β and Gas6 treatment. After treatment with 400 ng / ml Gas6 for 20 hours, 10 ng / ml of TGF-β was treated for 30 minutes or 1 hour. * P <0.05, relative to control.
8 shows the results of confirming phosphorylation of ERK1 / 2 protein in LA-4 cells following TGF-β and Gas6 treatment. After treatment with 400 ng / ml Gas6 for 20 hours, 10 ng / ml of TGF-β was treated for 5 minutes, 30 minutes or 1 hour. * P <0.05 compared to control; + P <0.05, compare displayed values.
Figure 9 shows the results confirming the phosphorylation of AKT protein in LA-4 cells following TGF-β and Gas6 treatment. After treatment with 400 ng / ml Gas6 for 20 hours, 10 ng / ml of TGF-β was treated for 1 hour, 3 hours or 8 hours. * P <0.05 compared to control; + P <0.05, compare displayed values.
Figure 10 shows the results confirming the phosphorylation of P38 protein in LA-4 cells following TGF-β and Gas6 treatment. After treatment with 400 ng / ml Gas6 for 20 hours, 10 ng / ml of TGF-β was treated for 15 minutes, 1 hour or 6 hours. * P <0.05, relative to control.
11 shows the results of confirming COX-2 or COX-1 mRNA levels in LA-4 cells following Gas6 treatment. 400 ng / ml Gas6 was treated at the indicated times, respectively. * P <0.05, relative to control.
12 shows the results of confirming COX-2 or COX-1 protein levels in LA-4 cells following Gas6 treatment. 400 ng / ml Gas6 was treated at the indicated times, respectively. * P <0.05, relative to control.
Figure 13 shows the results of confirming the PGE ₂ or PGD ₂ protein level in LA-4 cell culture medium following Gas6 treatment. 400 ng / ml Gas6 was treated for 8 or 20 hours. * P <0.05, relative to control.
14 shows the results of confirming the level of COX-2 protein in LA-4 cells following COX-2 specific siRNA treatment. LA-4 cells were transfected with COX-2 specific siRNA or control siRNA for 6 hours. * P <0.05, relative to control.
15 shows the results of confirming PGE ₂ or PGD ₂ protein levels in LA-4 cell culture medium following COX-2 specific siRNA treatment. LA-4 cells were transfected with COX-2 specific siRNA or control siRNA for 6 hours and then treated with 400 ng / ml Gas6 for 20 hours. * P <0.05 compared to control; + P <0.05, compare displayed values
Figure 16 shows the results of confirming the morphological changes of LA-4 cells following NS-398 treatment. LA-4 cells were treated with 10 μM NS-398 for 1 hour and 400 ng / ml Gas6 for 20 hours, and then stimulated with 10 ng / ml TGF-β for 72 hours.
Figure 17 shows the results of confirming the mRNA level of the EMT markers in LA-4 cells following NS-398 treatment. LA-4 cells were treated with 10 μM NS-398 for 1 hour and 400 ng / ml Gas6 for 20 hours, and then stimulated with 10 ng / ml TGF-β for 72 hours. * P <0.05 compared to control; + P <0.05, compare displayed values.
Figure 18 shows the results of confirming the protein level of the EMT markers in LA-4 cells following NS-398 treatment. LA-4 cells were treated with 10 μM NS-398 for 1 hour and 400 ng / ml Gas6 for 20 hours, and then stimulated with 10 ng / ml TGF-β for 72 hours. * P <0.05 compared to control; + P <0.05, compare displayed values.
19 shows the results of confirming the mRNA level of EMT markers in LA-4 cells following COX-2 specific siRNA treatment. LA-4 cells were transfected with COX-2 siRNA or control siRNA for 6 hours, treated with 400 ng / ml Gas6 for 20 hours, and then stimulated with 10 ng / ml TGF-β for 72 hours. * P <0.05 compared to control; + P <0.05, compare displayed values.
20 shows the results of confirming the protein levels of EMT markers in LA-4 cells following COX-2 specific siRNA treatment. LA-4 cells were transfected with COX-2 siRNA or control siRNA for 6 hours, treated with 400 ng / ml Gas6 for 20 hours, and then stimulated with 10 ng / ml TGF-β for 72 hours. * P <0.05 compared to control; + P <0.05, compare displayed values.
21 shows the results of confirming mRNA levels of Snai1, Zeb1 and Twist1 in LA-4 cells following NS-398 treatment. LA-4 cells were treated with 10 μM NS-398 for 1 hour and 400 ng / ml Gas6 for 20 hours, followed by 72 hours of stimulation with 10 ng / ml TGF-β. * P <0.05 compared to control; + P <0.05, compare displayed values.
22 shows the results of confirming mRNA levels of Snai1, Zeb1 and Twist1 in LA-4 cells following COX-2 siRNA treatment. LA-4 cells were treated with COX-2 siRNA or control siRNA for 6 hours and 400 ng / ml Gas6 for 20 hours, followed by 72 hours of stimulation with 10 ng / ml TGF-β. * P <0.05 compared to control; + P <0.05, compare displayed values.
Figure 23 shows the results of confirming the phosphorylation level of ERK1 / 2 protein in LA-4 cells following COX-2 siRNA treatment. LA-4 cells were treated with COX-2 siRNA or control siRNA for 6 hours and 400 ng / ml Gas6 for 20 hours, and then stimulated with 10 ng / ml TGF-β for 5 minutes. * P <0.05 compared to control; + P <0.05, compare displayed values.
24 shows the results of confirming the phosphorylation level of AKT protein in LA-4 cells following COX-2 siRNA treatment. LA-4 cells were treated with COX-2 siRNA or control siRNA for 6 hours and 400 ng / ml Gas6 for 20 hours, and then stimulated with 10 ng / ml TGF-β for 8 hours. * P <0.05 compared to control; + P <0.05, compare displayed values.
25 shows the results of confirming the change in the shape of LA-4 cells by the receptor antagonist treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours and 10 mM of antagonists EP2 (AH-6809), EP4 (AH-23848), DP1 (BW-A868C) or DP2 (BAY-u3405) of each receptor. Morphological changes were observed at 48 or 72 hours with or without TGF-β.
Figure 26 shows the results of confirming the mRNA level of EMT markers in LA-4 cells following receptor antagonist treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours, and treated with TGF-β for 48 hours under or without 10 mM of antagonist EP2 (AH-6809) or EP4 (AH-23848) of each receptor. It was. * P <0.05 compared to control; + P <0.05, compare displayed values.
Figure 27 shows the results of confirming the mRNA level of the EMT markers in LA-4 cells following receptor antagonist treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours and TGF-β for 48 hours in the presence or absence of 10 mM DP2 antagonist (BAY-u3405). * P <0.05 compared to control; + P <0.05, compare displayed values.
Figure 28 shows the results of confirming the mRNA level of the EMT markers in LA-4 cells following receptor antagonist treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours and TGF-β for 72 hours in the presence or absence of 10 mM DP1 antagonist (BW-A868C). * P <0.05 compared to control; + P <0.05, compare displayed values.
29 shows the results of confirming the protein levels of EMT markers in LA-4 cells following receptor antagonist treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours, and treated with TGF-β for 48 hours under or without 10 mM of antagonist EP2 (AH-6809) or EP4 (AH-23848) of each receptor. It was. * P <0.05 compared to control; + P <0.05, compare displayed values.
Figure 30 shows the results of confirming the protein level of the EMT markers in LA-4 cells following receptor antagonist treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours and TGF-β1 for 48 hours in the presence or absence of 10 mM DP2 antagonist (BAY-u3405). * P <0.05 compared to control; + P <0.05, compare displayed values.
Figure 31 shows the results of confirming the protein level of the EMT markers in LA-4 cells following receptor antagonist treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours and TGF-β for 72 hours in the presence or absence of 10 mM DP1 antagonist (BW-A868C). * P <0.05 compared to control; + P <0.05, compare displayed values.
32 shows the results of confirming Snai1, Zeb1 and Twist1 mRNA levels in LA-4 cells following receptor antagonist treatment. LA-4 cells treated with 400 ng / ml Gas6 for 20 hours, with or without 10 mM of antagonists EP2 (AH-6809), EP4 (AH-23848) or DP2 (BAY-u3405) of each receptor. TGF-β was treated for 48 hours. * P <0.05 compared to control; + P <0.05, compare displayed values.
33 shows the results of confirming Snai1, Zeb1, and Twist1 mRNA levels in LA-4 cells following receptor antagonist treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours and TGF-β for 72 hours in the presence or absence of 10 mM DP1 antagonist (BW-A868C). * P <0.05 compared to control; + P <0.05, compare displayed values.
34 shows the results of confirming HGF mRNA levels in LA-4 cells following Gas6 treatment. LA-4 cells were treated with 400 ng / ml Gas6 at the indicated times. * P <0.05, relative to control.
35 shows the results of confirming the HGF protein level in LA-4 cell culture medium following Gas6 treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 8 hours or 20 hours. * P <0.05, relative to control.
36 shows the results of confirming HGF protein levels in LA-4 cells following Gas6 treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 8 hours or 20 hours. * P <0.05, relative to control.
37 shows the results of confirming the level of RhoA protein in LA-4 cells following RhoA specific siRNA treatment. LA-4 cells were treated with RhoA specific siRNA or control siRNA for 24 hours. * P <0.05, relative to control.
38 shows the results of confirming EMT marker mRNA levels in LA-4 cells following RhoA specific siRNA treatment. LA-4 cells were treated with RhoA specific siRNA or control siRNA for 24 hours, and then treated with 400 ng / ml Gas6 for 20 hours and then stimulated with 10 ng / ml TGF-β for 48 hours. * P <0.05 compared to control; + P <0.05, compare displayed values.
39 shows the results of confirming the level of EMT marker protein in LA-4 cells following RhoA-specific siRNA treatment. LA-4 cells were treated with RhoA specific siRNA or control siRNA for 24 hours, and then treated with 400 ng / ml Gas6 for 20 hours and then stimulated with 10 ng / ml TGF-β for 48 hours. * P <0.05 compared to control; + P <0.05, compare displayed values.
40 shows the results of confirming the morphological changes of LA-4 cells following Y-27632 treatment. LA-4 cells were treated with 10 mM Y-27632 for 1 hour and then treated with 400 ng / ml Gas6 for 20 hours, and then stimulated with 10 ng / ml TGF-β for 48 hours. The following cell morphology changes were observed.
41 shows the results of confirming the level of EMT marker mRNA in LA-4 cells following Y-27632 treatment. LA-4 cells were treated with 10 mM Y-27632 for 1 hour and then treated with 400 ng / ml Gas6 for 20 hours and then stimulated with 10 ng / ml TGF-β for 48 hours. * P <0.05 compared to control; + P <0.05, compare displayed values.
42 shows the results of confirming the level of EMT marker protein in LA-4 cells following Y-27632 treatment. LA-4 cells were treated with 10 mM Y-27632 for 1 hour and then treated with 400 ng / ml Gas6 for 20 hours, and then stimulated with 10 ng / ml TGF-β for 48 hours. * P <0.05 compared to control; + P <0.05, compare displayed values.
43 shows the results of confirming Snai1, Zeb1 and Twist1 mRNA levels in LA-4 cells following RhoA specific siRNA treatment. LA-4 cells were transfected with RhoA siRNA or control siRNA for 24 hours, treated with 400 ng / ml Gas6 for 20 hours, and then stimulated with 10 ng / ml TGF-β for 72 hours. * P <0.05 compared to control; + P <0.05, compare displayed values.
44 shows the results of confirming Snai1, Zeb1 and Twist1 mRNA levels in LA-4 cells following Y-27632 treatment. LA-4 cells were treated with 10 mM Y-27632 for 1 hour, treated with 400 ng / ml Gas6 for 20 hours, and then stimulated with 10 ng / ml TGF-β for 48 hours. * P <0.05 compared to control; + P <0.05, compare displayed values.
45 shows the results of confirming the phosphorylation level of ERK1 / 2 protein in LA-4 cells following RhoA specific siRNA treatment. LA-4 cells were transfected with RhoA siRNA or control siRNA for 24 hours, treated with 400 ng / ml Gas6 for 20 hours, and then stimulated with 10 ng / ml TGF-β for 5 minutes. * P <0.05 compared to control; + P <0.05, compare displayed values.
46 shows the results of confirming the phosphorylation level of AKT protein in LA-4 cells following RhoA specific siRNA treatment. LA-4 cells were transfected with RhoA siRNA or control siRNA for 24 hours, treated with 400 ng / ml Gas6 for 20 hours, and then stimulated with 10 ng / ml TGF-β for 8 hours. * P <0.05 compared to control; + P <0.05, compare displayed values.
Fig. 47 shows the results of confirming the morphology of LA-4 cells following c-Met antagonist treatment. After treating LA-4 cells with 400 ng / ml Gas6 for 20 hours, TGF-β was treated for 72 hours in the presence or absence of 250 nM of the c-Met antagonist PHA-665752, and morphological changes were observed.
48 shows the results of confirming the mRNA levels of EMT markers in LA-4 cells following c-Met antagonist treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours and then treated with TGF-β for 72 hours in the presence or absence of 250 nM of PHA-665752, a c-Met antagonist. * P <0.05 compared to control; + P <0.05, compare displayed values.
Figure 49 shows the results of confirming the protein level of the EMT markers in LA-4 cells following c-Met antagonist treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours and then treated with TGF-β for 72 hours in the presence or absence of 250 nM of PHA-665752, a c-Met antagonist. * P <0.05 compared to control; + P <0.05, compare displayed values.
50 shows the results of confirming mRNA levels of Snai1, Zeb1, and Twist1 in LA-4 cells following c-Met antagonist treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours and then treated with TGF-β for 72 hours in the presence or absence of 250 nM of PHA-665752, a c-Met antagonist. * P <0.05 compared to control; + P <0.05, compare displayed values.
Figure 51 shows the results of confirming the protein level of the EMT markers in LA-4 cells following PGE ₂ , PGD ₂ or HGF treatment. LA-4 cells were treated with PGE ₂ (35 or 118 pg / ml), PGD ₂ (6 or 28 pg / ml) or HGF (169 or 194 pg / ml) with 10 ng / ml TGF-β for 72 hours. . * P <0.05 compared to control; + P <0.05, compare displayed values.
Figure 52 shows the results of confirming the protein level of the EMT markers in LA-4 cells following HGF treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours and then replaced with fresh medium, followed by 72 hours of treatment with HGF (169 or 194 pg / ml) with 10 ng / ml TGF-β. * P <0.05 compared to control; + P <0.05, compare displayed values.
FIG. 53 shows the results of confirming c-Met, EP2, EP4, DP1 or DP2 receptor protein levels in LA-4 cells following Gas6 treatment. FIG. LA-4 cells were treated with 400 ng / ml Gas6 for 12 hours or 20 hours. * P <0.05 compared to control; + P <0.05, compare displayed values.
54 shows the results of confirming Axl or Mer expression levels in LA-4 cells following Gas6 treatment. 400 ng / ml Gas 6 was treated for 0, 5, 15, 30, 60 and 120 minutes. * P <0.05 compared to control; + P <0.05, compare displayed values.
55 shows the results of confirming Axl or Mer protein expression levels in LA-4 cells following Axl or Mer specific siRNA treatment. LA-4 cells were treated with Axl or Mer siRNA or control siRNA for 48 hours and treated with 400 ng / ml Gas6. * P <0.05 compared to control; + P <0.05, compare displayed values.
FIG. 56 shows the results of confirming COX-2 mRNA, PGE ₂ and PGD ₂ expression levels in LA-4 cells following Axl or Mer specific siRNA treatment. FIG. LA-4 cells were treated with Axl or Mer siRNA or control siRNA for 48 hours and treated with 400 ng / ml Gas6. * P <0.05 compared to control; + P <0.05, compare displayed values.
57 shows the results of confirming RhoA activity, HGF mRAN and protein expression levels in LA-4 cells following Axl or Mer specific siRNA treatment. LA-4 cells were treated with Axl or Mer siRNA or control siRNA for 48 hours and treated with 400 ng / ml Gas6. * P <0.05 compared to control; + P <0.05, compare displayed values.
58 shows the results of confirming the mRNA levels of EMT markers in LA-4 cells following Axl or Mer specific siRNA treatment. LA-4 cells were treated with Axl or Mer siRNA or control siRNA for 48 hours, treated with 400 ng / ml Gas6 for 20 hours, and then treated with TGF-β for 72 hours. * P <0.05 compared to control; + P <0.05, compare displayed values.
Figure 59 shows the results of confirming the protein expression level of EMT markers in LA-4 cells by Axl or Mer specific siRNA treatment. LA-4 cells were treated with Axl or Mer siRNA or control siRNA for 48 hours, treated with 400 ng / ml Gas6 for 20 hours, and then treated with TGF-β for 72 hours. * P <0.05 compared to control; + P <0.05, compare displayed values.
60 shows the results of confirming mRNA levels of Snai1, Zeb1, and Twist1 in LA-4 cells following Axl or Mer specific siRNA treatment. LA-4 cells were treated with Axl or Mer siRNA or control siRNA for 48 hours, treated with 400 ng / ml Gas6 for 20 hours, and then treated with TGF-β for 72 hours. * P <0.05 compared to control; + P <0.05, compare displayed values.
Figure 61 shows the results confirming the phosphorylation of ERK1 / 2 protein in LA-4 cells by Axl or Mer specific siRNA treatment. LA-4 cells were treated with Axl or Mer siRNA or control siRNA for 48 hours, treated with 400 ng / ml Gas6 for 20 hours, and then treated with TGF-β for 72 hours. * P <0.05 compared to control; + P <0.05, compare displayed values.
Figure 62 shows the results confirming the phosphorylation of AKT protein in LA-4 cells by Axl or Mer specific siRNA treatment. LA-4 cells were treated with Axl or Mer siRNA or control siRNA for 48 hours, treated with 400 ng / ml Gas6 for 20 hours, and then treated with TGF-β for 72 hours. * P <0.05 compared to control; + P <0.05, compare displayed values.
63 shows the results of confirming the expression level of EMT marker mRNA in primary mouse AT II cells following Gas6 treatment. After treatment with 400 ng / ml Gas6 for 20 hours, TGF-β was treated for 48 or 72 hours. * P <0.05 compared to control; + P <0.05, compare displayed values.
64 shows the results of confirming the expression level of EMT marker protein in primary mouse AT II cells following Gas6 treatment. After treatment with 400 ng / ml Gas6 for 20 hours, TGF-β was treated for 72 hours. * P <0.05 compared to control; + P <0.05, compare displayed values.
65 shows the results of confirming the expression levels of Snai1 / 2, Zeb1 / 2 and Twist1 mRNA in primary mouse AT II cells following TGF-β and Gas6 treatment. After treatment with 400 ng / ml Gas6 for 20 hours, 10 ng / ml of TGF-β was treated for 48 hours or 72 hours. * P <0.05 compared to control; + P <0.05, compare displayed values.
66 shows the results of confirming the level of COX-2 mRNA in primary mouse AT II cells following Gas6 treatment. 400 ng / ml Gas 6 was each treated at the indicated times. * P <0.05, relative to control.
67 shows the results of confirming the expression level of EMT marker mRNA in primary mouse AT II cells following NS-398 and Gas6 treatment. After 1 hour treatment with 10 μM NS-398, 400 ng / ml Gas6 was treated for 20 hours followed by 72 hours of 10 ng / ml TGF-β. * P <0.05 compared to control; + P <0.05, compare displayed values.
68 shows the results of confirming the expression levels of Snai1 / 2, Zeb1 / 2 and Twist1 mRNA in primary mouse AT II cells following NS-398 and Gas6 treatment. After 1 hour treatment with 10 μM NS-398, 400 ng / ml Gas6 was treated for 20 hours followed by 72 hours of 10 ng / ml TGF-β. * P <0.05 compared to control; + P <0.05, compare displayed values.
69 shows the results of confirming the expression level of EMT marker mRNA in primary mouse AT II cells following AH6, BAY and Gas6 treatment. After treatment with 400 ng / ml Gas6 for 20 hours, only TGF-β was treated for 48 hours, 10 μM of EP2 (AH-6809) or 10 μM of DP2 (BAY-u3405). * P <0.05 compared to control; + P <0.05, compare displayed values.
70 shows the results of confirming the expression levels of Snai1 / 2, Zeb1 / 2 and Twist1 mRNA in primary mouse AT II cells following AH6, BAY and Gas6 treatment. After treatment with 400 ng / ml Gas6 for 20 hours, only TGF-β was treated for 48 hours, 10 μM of EP2 (AH-6809) or 10 μM of DP2 (BAY-u3405). * P <0.05 compared to control; + P <0.05, compare displayed values.
71 shows the results of confirming HGF mRNA levels in primary mouse AT II cells following Gas6 treatment. LA-4 cells were treated with 400 ng / ml Gas6 at the indicated times. * P <0.05, relative to control.
72 shows the results of confirming primary mouse AT II cell EMT marker mRNA levels following Y-27632 treatment. LA-4 cells were treated with 10 mM Y-27632 for 1 hour and then treated with 400 ng / ml Gas6 for 20 hours and then stimulated with 10 ng / ml TGF-β for 48 hours. * P <0.05 compared to control; + P <0.05, compare displayed values.
73 shows the results of confirming the primary mouse AT II cells Snai1, Zeb1 and Twist1 mRNA levels following Y-27632 treatment. LA-4 cells were treated with 10 mM Y-27632 for 1 hour and then treated with 400 ng / ml Gas6 for 20 hours and then stimulated with 10 ng / ml TGF-β for 48 hours. * P <0.05 compared to control; + P <0.05, compare displayed values.
74 shows the results of confirming the mRNA levels of EMT markers in primary mouse AT II cells following c-Met antagonist treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours and then treated with TGF-β for 72 hours in the presence or absence of 250 nM of PHA-665752, a c-Met antagonist. * P <0.05 compared to control; + P <0.05, compare displayed values.
75 shows the results of confirming Snai1, Zeb1, and Twist1 mRNA levels in primary mouse AT II cells following c-Met antagonist treatment. LA-4 cells were treated with 400 ng / ml Gas6 for 20 hours and then treated with TGF-β for 72 hours in the presence or absence of 250 nM of PHA-665752, a c-Met antagonist. * P <0.05 compared to control; + P <0.05, compare displayed values.
76 shows human lung adenocarcinoma cells (549 cells) following TGF-β and Gas6 treatment. The result of confirming the expression level of EMT marker mRNA in this is shown. After treatment with 400 ng / ml Gas6 for 20 hours, 10 ng / ml of TGF-β was treated for 48 hours or 72 hours. * P <0.05 compared to control; + P <0.05, compare displayed values.
FIG. 77 shows the results of confirming the expression level of EMT marker protein in human lung adenocarcinoma cells (549 cells) following TGF-β and Gas6 treatment. After treatment with 400 ng / ml Gas6 for 20 hours, 10 ng / ml of TGF-β was treated for 48 hours or 72 hours. * P <0.05 compared to control; + P <0.05, compare displayed values.
78 shows the results of confirming the levels of Snai1 / 2, Zeb1 / 2 and Twist1 mRNA in human lung adenocarcinoma cells (549 cells) following TGF-β and Gas6 treatment. After treatment with 400 ng / ml Gas6 for 20 hours, 10 ng / ml of TGF-β was treated for 48 hours or 72 hours. * P <0.05 compared to control; + P <0.05, compare displayed values.
FIG. 79 shows the results of confirming the shape of primary mouse AT II cells following in vivo bleomycin and Gas6 treatment by phase contrast microscope. FIG.
80 shows the results of confirming the expression level of EMT marker mRNA in primary mouse AT II cells following in vivo bleomycin and Gas6 treatment. * P <0.05 compared to control; + P <0.05, compare displayed values.
Figure 81 shows the results of confirming the expression level of Snai1, Zeb1 and Twist1 mRNA of primary mouse AT II cells following treatment with bleomycin and Gas6 in vivo. * P <0.05 compared to control; + P <0.05, compare displayed values.
FIG. 82 shows the results of confirming COX-2 mRNA levels of primary mouse AT II cells following in vivo bleomycin and Gas6 treatment. * P <0.05 compared to control; + P <0.05, compare displayed values.
FIG. 83 shows the results of confirming PGE ₂ or PGD ₂ protein levels in primary mouse AT II cells following in vivo bleomycin and Gas6 treatment. * P <0.05 compared to control; + P <0.05, compare displayed values.
FIG. 84 shows the results of confirming HGF mRNA levels of primary mouse AT II cells following in vivo bleomycin and Gas6 treatment. * P <0.05 compared to control; + P <0.05, compare displayed values.
FIG. 85 shows the results of confirming HGF and TGF-β protein levels of primary mouse AT II cells following in vivo bleomycin and Gas6 treatment. * P <0.05 compared to control; + P <0.05, compare displayed values.
86 shows the results of confirming the expression levels of EMT markers and extracellular matrix proteins in lung tissues on day 14 following in vivo bleomycin and Gas6 treatment. * P <0.05 compared to control; + P <0.05, compare displayed values.
FIG. 87 shows the results of confirming the expression levels of EMT markers and extracellular matrix proteins in lung tissues at 21 days following in vivo bleomycin and Gas6 treatment. * P <0.05 compared to control; + P <0.05, compare displayed values.
FIG. 88 shows the results of confirming hydroxyproline levels in lung tissues at 21 days following in vivo bleomycin and Gas6 treatment. * P <0.05 compared to control; + P <0.05, compare displayed values.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1: Cell Lines and Cultures

LA-4 and HEK-293 cells were purchased from ATCC. LA-4 cells were cultured in F12K medium (Lonza, Switzerland) containing 15% FBS (fetal bovine serum) inactivated by heat treatment at 37 ℃ 5% CO ₂ conditions. HEK-293 cells contain DMEM (Dulbecco's modified Eagle's medium; Media Media, USA) containing 10% FBS, 2 mM L-glutamine, 100 U / ml penicillin and 100 mg / ml streptomycin at 37 ° C. 5% CO ₂ conditions. Incubated at.

Example 2: Isolation and Culture of Primary Cells

Primary mouse AT II (alveolar type II) epithelial cells were isolated and purified from BALB / c mice.

Pulmonary blood was removed by injection of 0.9% saline into the pulmonary artery. After washing the lungs with 1 ml saline, 100 units of dispase were injected into the mouse lungs, and then incubated for 45 minutes at room temperature. The lungs were then isolated from large bronchus by mechanical means, and the isolated lung tissues were incubated for 10 minutes at 37 ° C. in DMEM medium containing 0.01% of DNase I. Cells were filtered, centrifuged and resuspended in sequential plating in cell culture Petri dishes coated with mouse IgG 0.75 mg / ml for 1 hour at 37 ° C. to remove macrophages and fibroblasts, respectively. Final cell isolates were prepared with Ham's F12 medium containing 15 mM HEPES, 0.8 mM CaCl ₂ , 0.25% BSA, 5 mg / ml insulin, 5 mg / ml transferrin, 5 ng / ml sodium selenite and 2% mouse serum. Was inoculated into a type 1 collagen coated 35-mm petri dish added thereto.

Purity of AT II cells assessed by pro-SP-C immunofluorescence staining was greater than 90%.

Example 3: Incubation of Epithelial Cells

LA-4 and HEK-293 cells were seeded in 6-well culture dishes (2 × 10 ⁵ cells / well) and incubated overnight in 200 μl of RPMI 1640 or DMEM containing 10% FBS. Primary AT II cells were seeded in type 1 collagen coated culture dishes (1 × 10 ⁶ cells / well) and incubated for 48 hours. Cells were treated with 400 ng / ml of Gas6 for 20 hours in the presence or absence of 10 ng / ml of TGF-β (R & D Systems Inc). In some experiments 10 μM NS-398 for COX-2 inhibition, 30 mM Y-27632 was used for Rho kinase inhibition. Each inhibitor was added 1 hour before Gas6 treatment. In addition, AH-6809, AH-23848, BW-A868C, BAY-u3405 10 mM or PHA-665752 250 nM were used as antagonists for EP2, EP4, DP1, DP2 or c-Met, respectively. Each receptor antagonist was added 1 hour before TGF-β treatment.

Example 4: transient transfection

5 μl of siRNA transfection reagent (Genlantis) was used to transiently transfect LA-4 cells with COX-2 or RhoA specific siRNA, or 1 μg / ml of control siRNA. Axl or Mer specific siRNA used 75 nM. The siRNA sequences used are shown in Table 1.

siRNA target Sense sequence Antisense sequence COX-2 5'-CUAUGAUAGGAGCAUGUAA-3 '
(SEQ ID NO 1) 5'-UUACAUGCUCCUAUCAUAG-3 '
(SEQ ID NO: 2) RhoA 5'-GAAGUCAAGCAUUUCUGUCUUA-3 '
(SEQ ID NO: 3) 5'-GACAGAAAUGCUUGACUUCUU-3 '
(SEQ ID NO: 4) Axl 5′-GAGAUGGACAGAUCCUAGA-3 ′
(SEQ ID NO: 5) 5′-UCUAGGAUCUGUCCAUCUC-3 ′
(SEQ ID NO: 6) Mer 5′-CACAGUUUUAUCCUGAUGA-3 '
(SEQ ID NO: 7) 5′-UCAUCAGGAUAAAACUGUG-3 ′
(SEQ ID NO: 8) Control 5'-CCUACGCCACCAAUUUCGU-3 '
(SEQ ID NO: 9) 5'-ACGAAAUUGGUGGCGUAGG-3 '
(SEQ ID NO: 10)

Cells were cultured for 6 hours in serum-free medium containing COX-2 siRNA, 24 hours in serum-free medium containing RhoA siRNA, or 48 hours in serum-free medium containing Axl or Mer siRNA. No significant effect on cell viability was observed for all siRNAs used.

Example 5: Immunoblot Analysis

To confirm epithelial marker and mesenchymal marker expression, cells were lysed in lysis buffer containing 0.5% Triton X-100, loaded onto 10% SDS-PAGE gel and transferred to nitrocellulose membrane. The membrane was blocked with Tris-buffered saline (TBS) containing 3% BSA or 5% skim milk at room temperature, then incubated with each anti-mouse primary antibody at room temperature and attached to an anti-mouse HRP-binding secondary antibody. . Protein bands were identified using enhanced chemiluminescence.

Example 6: Enzyme-linked immunosorbent assay (ELISA) measurement

The cell culture supernatant was recovered and subjected to ELISA. PGE ₂ PGD ₂ , HGF and TGF-β concentration levels of active forms were measured with an ELISA kit (R & D Systems, Minneapolis, MN).

Example 7: Quantitative real-time PCR (qPCR)

Gene expression was analyzed by real-time qPCR in a StepOnePlus system (Applied Biosystems, Life Technologies, Carlsbad, Calif., USA). In each qPCR analysis, a total of 50 ng cDNA was used. Primer Express software was used to design primer sets for PCR-based amplification. Primers used in the present invention are shown in Table 2.

primer Forward Reverse E-cadherin 5'-GCACTCTTCTCCTGGTCCTG-3 '
(SEQ ID NO: 11) 5'-TATGAGGCTGTGGGTTCCTC-3 '
(SEQ ID NO: 12) N-cadherin 5'-CCTCCAGAGTTTACTGCCATGAC-3 '
(SEQ ID NO: 13) 5'-CCACCACTGATTCTGTATGCCG-3 '
(SEQ ID NO: 14) α-SMA 5'-CCACCGCAAATGCTTCTAAGT-3 '
(SEQ ID NO: 15) 5'-GGCAGGAATGATTTGGAAAGG-3 '
(SEQ ID NO: 16) Snai1 5'-CCCAAGGCCGTAGAGCTGA-3 '
(SEQ ID NO: 17) 5'-GCTTTTGCCACTGTCCTCATC-3 '
(SEQ ID NO: 18) Snai2 5'-ATCCTCACCTCGGGAGCATA-3 '
(SEQ ID NO: 19) 5'-TGCCGACGATGTCCATACAG-3 '
(SEQ ID NO: 20) Zeb1 5'-ATTCAGCTACTGTGAGCCCTGC-3 '
(SEQ ID NO: 21) 5'-CATTCTGGTCCTCCACAGTGGA-3 '
(SEQ ID NO 22) Zeb2 5'-GCAGTGAGCATCGAAGAGTACC-3 '
(SEQ ID NO: 23) 5'-GGCAAAAGCATCTGGAGTTCCAG-3 '
(SEQ ID NO: 24) Twist1 5'-TCGACTTCCTGTACCAGGTCCT-3 '
(SEQ ID NO: 25) 5'-CCATCTTGGAGTCCAGCTCG-3 '
(SEQ ID NO 26) COX-1 5'-CGATCTGGCTTCGTGAAC-3 '
(SEQ ID NO 27) 5'-GAGCTGCAGGAAATAGCC-3 '
(SEQ ID NO 28) COX-2 5'-GGGAGTCTGGAACATTGTGA-3 '
(SEQ ID NO 29) 5'-GTGCACATTGTAAGTAGGTG-3 '
(SEQ ID NO: 30) HGF 5'-CACCCCTTGGGAGTATTGTG-3 '
(SEQ ID NO 31) 5'-GGGACATCAGTCTCATTCAC-3 '
(SEQ ID NO: 32) HPRT 5'-CCAGTGTCAATTATATCTTCAAC-3 '
(SEQ ID NO: 33) 5'-CAGACTGAAGAGCTACTGTAATG-3 '
(SEQ ID NO 34)

cDNA was normalized to the amount of hypoxanthine-guanine phosphoribosyltransferase (HPRT) and expressed as a fold-change compared to the control.

Example 8: RhoA Activity Assay

RhoA activity was measured in LA-4 cell lysates using an ELISA-based RhoA activation assay Biochem Kit (G-LISA; Cytoskeleton). Cell lysates were added to RhoA-GTP affinity plates coated with Rhotekin binding domains of RhoA for 30 minutes. The active GTP-binding form of RhoA was measured using indirect immunodetection and chromatic response was measured at 490 nm with a microplate spectrophotometer.

Example 9: Hydroxyproline Measurement

The hydroxyproline content was measured using a hydroxyproline assay kit (Jiancheng Bioengineering, Nanjing, China) according to the manufacturer's instructions.

Example 10 Animal Experiment

All experiments used 20-25 g of specific pathogen-free male C57BL / 6 mice (Orient Bio, Seongnam, Korea). Mouse pharyngeal aspiration was administered a test solution containing bleomycin (30 μl of 5 U / kg body weight). Saline alone or Gas6 (50 μg / kg, i.p.) was administered one day before bleomycin administration. Inhibitors were administered once a day after the first bleomycin dosing and mice were euthanized on days 14 and 21 of bleomycin administration.

Example 11: Statistical Analysis

Mean values are expressed as ± SEM. ANOVA was applied for various analyzes, and Tukey's post hoc test was applied. Student's t-tests were used to compare the sample mean of the two samples. P values less than 0.05 were considered statistically significant. All data were analyzed using JMP software (SAS Institute, Cary, NC).

Experimental Example 1: Inhibitory effect of Gas6 on TGF-β-induced EMT in lung and kidney epithelial cells

TGF-β signaling is known to play an important role in the development of EMT and fibrosis (Am J Physiol Lung Cell Mol Physiol, 2007, 293: L525). We sought to determine whether Gas6 treatment modulates the TGF-β-induced EMT process in mouse alveolar type 2 pulmonary epithelial cells (LA-4).

First, it was confirmed that LA-4 cells were converted to spindle-like morphology when stimulated with 48 hours or 72 hours by 10 ng / ml TGF-β treatment, but 400 ng with TGF-β. Treatment of / 6 with Gas6 prevented spindle-like morphology conversion of LA-4 cells and confirmed morphology of epithelial cells (FIG. 1). In addition, stimulation of 48 ng or 72 hrs with 10 ng / ml TGF-β resulted in decreased expression of the adherens junction protein, E-cadherin, and the expression of myofibroblast differentiation markers N-cadherin and α-SMA (α- Although smooth muscle actin) expression was increased, it was confirmed at the protein and mRNA levels that such expression of EMT markers was suppressed when 400 ng / ml of Gas6 was treated with TGF-β (FIGS. 2 and 3). The data suggest that biosynthetic mediators synthesized and secreted after Gas6 treatment could block TGF-β signals in autocrine fashion in LA-4 cells. In addition, the HEK-293 human kidney epithelial cells (FIG. 4) confirmed that the TGF-β-induced EMT marker change was inhibited at the protein level through Gas6 treatment.

Experimental Example 2: Effect of Gas6 Treatment on Smad-Independent Signaling and EMT Regulatory Transcription Factor Expression Induced by TGF-β in LA-4 Cells

In EMT induction, activated Smad or non-Smad signaling mediates transcriptional regulation of Snai, Zeb, and Basic helix-loop-helix transcription factor families, inhibiting epithelial marker gene expression, and mesenchymal genes. (mesenchymal gene) activates expression (Sci Signal, 2014, 7: re8). Thus, the present inventors attempted to determine whether Gas6 inhibits the expression of the transcription factor in LA-4 cells stimulated with TGF-β.

Treatment of TGF-β with TGF-β 20 hours after treatment with Gas6 on LA-4 cells or HEK 293 cells confirmed that all mRNA expressions of Snai1 / 2, Zeb1 / 2 and Twist1 induced by TGF-β were inhibited. (FIGS. 5 and 6, respectively). Treatment of Gas6 in LA-4 cells did not affect the phosphorylation of Smad2 and Smad3 mediated by TGF-β (FIG. 7), but phosphorylation of extracellular signal-regulated kinase (ERK) and Akt induced by TGF-β Was partially inhibited (FIGS. 8 and 9). There was no effect on phosphorylation of p38 mitogen-activated protein kinase (FIG. 10). The data suggest that bioactive mediators synthesized or secreted after Gas6 treatment substantially block Smad-independent TGF-β signals, including the ERK and Akt pathways, thereby preventing the transcriptional regulators from blocking EMT in LA-4 cells. Suggests that it decreases.

Experimental Example 3: COX-2 Derived PGE by Gas6 Treatment in LA-4 Cells ₂  And PGD ₂  Secretion, and its effect on inhibiting EMT

COX-2 / PGE ₂ and PGD ₂ pathways are known to inhibit EMT in lung and kidney epithelial cells (Sci Rep, 2016, 6: 20992). Therefore, the present inventors attempted to determine whether COX-2-derived PGE ₂ and PGD ₂ secreted from LA-4 in response to Gas6 mediate anti-EMT effects in LA-4 cells by self-secretion.

First, the present inventors confirmed the mRNA and protein expression of COX-1 and COX- ₂ and the production of PGE ₂ and PGD ₂ by Gas6 in LA-4 cells. As a result, COX-2 mRNA levels showed a maximum after 1 hour of Gas6 treatment, returned to the original level at 20 hours, and COX-1 mRNA expression did not change within 24 hours after Gas6 treatment (FIG. 11). COX-2 protein expression gradually increased up to 24 hours after Gas6 treatment, but COX-1 expression did not change during this period (FIG. 12). In addition, 20 hours after Gas6 treatment, PIA ₂ and PGD ₂ secretion measured by EIA in LA-4 cells increased 3.3 and 2.5 fold, respectively (FIG. 13).

Next, to confirm that COX-2 induction by Gas6 stimulation in LA-4 cells improves PGE ₂ and PGD ₂ production, LA-4 cells are transfected with COX-2 specific siRNA or negative-control siRNA And incubated for 6 hours. As a result, the negative-control siRNA did not affect the amount of COX-2 protein in the cells, but in cells transfected with COX-2 specific siRNA, the amount of COX-2 protein was ~ 60% or more compared to wild-type LA-4 cells. Decreased (FIG. 14). Transfection of COX-2 siRNA prevented Gas6-induced PGE ₂ and PGD ₂ secretion (FIG. 15), suggesting that increased gas _6- induced PGE ₂ and PGD ₂ production in LA-4 cells resulted in COX-2 expression. Suggests that it is derived from induction.

In order to further confirm the functional correlation of COX-2 signal in the anti-EMT effect of Gas6, LA-3 cells were treated with NS-398, a highly selective COX-2 inhibitor, for 1 hour before Gas6 treatment. As a result, NS-398 reversed all of the effects of Gas6 on TGF-β-induced cell morphology changes and E-cadherin loss and the synthesis of α-SMA and N-cadherin at the gene and protein levels (Figs. 16 to 16). 18). In addition, when knocking down the COX-2 gene in LA-4 cells, the effect of Gas6 was similarly reversed (FIGS. 19 and 20). Furthermore, both NS-398 and COX-2 siRNA reversed the effect of Gas6 on reducing TGF-β-induced Snai1, Zeb1 and Twist1 mRNA expression (FIGS. 21 and 22). In addition, COX-2 siRNA reversed the effect of Gas6 on reducing TGF-β-induced phosphorylation of ERK1 / 2 and AKT in LA-4 cells, but did not show this result in negative control siRNA (FIGS. 23 and 24). ).

Next, to determine whether PGE ₂ and PGD ₂ secretion derived from COX-2 mediates anti-EMT effects in LA-4 cells, LA-4 cells were treated one hour before TGF-β treatment in the presence or absence of Gas6. PGE ₂ -or PGD ₂ -specific, such as E-prostanoid-2 receptor antagonist (AH-6809), EP4 antagonist (AH-23848), DP1 antagonist (BW-A868C) or DP2 antagonist (BAY-u3405) The antagonists of the receptors were treated. As a result, the anti-EMT effect of Gas6 was significantly reversed by the antagonists of EP2 and DP2 (FIGS. 25-27, 29, 30 and 32). However, EP4 antagonists and DP1 antagonists weakly or had little effect on the anti-EMT effect of Gas6 (FIGS. 25, 26, 28, 29, 31 and 33). The results suggest that anti-EMT effects in LA-4 cells are mediated primarily by COX-2 derived PGE ₂ and PGD ₂ signals, respectively, via EP2 and DP2 receptors.

Experimental Example 4: Confirmation of RhoA pathway-dependent HGF secretion and its EMT inhibitory effect by Gas6 treatment in LA-4 cells

We have previously reported that Gas6 induces mRNA and protein production of HGF in macrophages via a RhoA-dependent pathway (J Pharmacol Exp Ther, 2014, 350: 563). HGF mediates TGF-β-induced EMT in LA-4 cells (Sci Rep, 2016, 6: 20992). Therefore, the present inventors attempted to confirm the role of RhoA-dependent HGF secretion in LA-4 cells in Gas6 induced anti-EMT effect.

Gas6 treatment of LA-4 cells resulted in gas6-induced HGF mRNA expression at the highest level at 3 hours and then decreased to half level by 6 hours (FIG. 34). HGF secretion of LA-4 cells measured by ELISA was slightly increased due to Gas6 treatment as compared to basal secretion levels (FIG. 35). Immunoblot analysis of LA-4 cell lysates using anti-HGF α-chain antibody showed that intracellular HGF protein levels were also increased by Gas6 treatment (FIG. 36).

Next, in order to inhibit RhoA / Rho kinase signal, LA-4 cells were transfected with RhoA-specific siRNA for 24 hours or treated with Rho kinase inhibitor Y-27632 for 1 hour before Gas6 stimulation. As a result, both RhoA knockdown and Rho kinase inhibition reversed the effect of Gas6 on TGF-β-induced EMT (FIGS. 37-44). RhoA siRNA also completely reversed Gas6's effect on phosphorylation of TGF-β-induced ERK1 / 2 and AKT in LA-4 cells, but the negative control did not show this effect (FIGS. 45 and 46). In addition, the anti-EMT effect of Gas6 was reversed even when c-Met signal was suppressed by treating LA-4 cells with PHA-665752, a c-Met antagonist 1 hour before TGF-β treatment (FIGS. 47 to 50). . The data strongly suggest that RhoA / Rho kinase-dependent HGF production in LA-4 cells also mediates the anti-EMT effect of Gas6 via c-Met signaling by autosecretion.

Experimental Example 5 Effect of Gas6 on EP2, DP2 and c-Met Expression Enhancement

As confirmed in the previous examples, PGE ₂ , PGD ₂ and HGF produced by Gas6 treatment mainly result in anti-EMT signals via EP2, DP2 and c-MET, respectively. To assess whether PGE ₂ , PGD ₂ and HGF act in an autosecretory manner as EMT inhibitors, the effect of the soluble mediator on LA-4 cells was determined at baseline concentrations (35, 6 and 169 pg / ml, respectively) and Stimulation concentrations were identified (118, 28 and 194 pg / ml, respectively).

As a result, stimulus concentrations of PGE ₂ and PGD ₂ inhibited changes in TMT-β-induced EMT markers at the protein level compared to basal concentrations (FIG. 51). The data support that EMT inhibition is significantly induced by Gas6 induced PGE ₂ and PGD ₂ release in LA-4 cells.

In contrast, HGF showed no anti-EMT effect at both stimulus concentration (194 pg / ml) and basal concentration (169 pg / ml) (FIG. 51), but after 20 hours of pretreatment with Gas6 and replacement with fresh medium HGF When 194 pg / ml was added, EGF marker changes induced by TGF-β were inhibited (FIG. 52). However, basal concentrations of HGF did not show anti-EMT effects under these experimental conditions. Furthermore, c-MET protein amount was increased 20 hours after Gas6 treatment (FIG. 53). This data suggests that despite the relatively low HGF production, HGF can have anti-EMT activity under these experimental conditions because it increases c-MET expression. Gas6 treatment in LA-4 cells also increased the amount of EP2 and DP2 protein, but did not increase the amount of EP4 and DP1 protein (FIG. 53). On the other hand, the addition of all of these mediators at stimulus concentrations in LA-4 cells showed no synergistic effect (FIG. 51), suggesting that they inhibit the signaling pathway induced by TGF-β through the same target molecule.

Experimental Example 6: Effect of Axl or Mer on Gas6-Induced EMT Inhibition in LA-4 Cells

Next, the present inventors attempted to determine whether Gas6 / Axl or Mer signals are regulated by Gas6 in LA-4 cells.

400 ng / ml of Gas6 was treated hourly (0, 5, 15, 30, 60 and 120 minutes) in LA-4 cells, and phosphorylation levels of Axl or Mer were measured (FIG. 54). As a result, both Axl or Mer phosphorylation levels peaked after 30 minutes of Gas6 treatment and gradually decreased.

Next, LA-4 cells were transfected with Axl or Mer specific siRNA or negative-control siRNA, incubated for 48 hours, and Axl or Mer expression levels were measured (FIG. 55). As a result, the negative-control siRNA did not affect the amount of Axl or Mer protein in cells, but in cells transfected with Axl or Mer specific siRNA, the amount of Axl or Mer protein was reduced by more than 70% compared to wild-type LA-4 cells. It became.

In addition, transfection of Axl or Mer siRNA prevented Gas6-induced COX-2 mRNA elevation, PGE ₂ and PGD ₂ secretion (FIG. 56), which was induced by Gas6-induced PGE ₂ and PGD in LA-4 cells. ₂ suggests that increased production results from induction of Axl or Mer expression.

In addition, transfection of Axl or Mer siRNA prevented the increase of Gasho-induced RhoA activity, HGF mRNA and protein levels (FIG. 57), which increased Gas6-induced RhoA activity in LA-4 cells, HGF mRNA And increased protein levels are derived from induction of Axl or Mer expression.

In addition, the present inventors attempted to confirm the effect of Axl or Mer on EMT inhibition by Gas6 in LA-4 cells.

LA-4 cells were transfected with Axl or Mer specific siRNA or negative-control siRNA, incubated for 48 hours, and mRNA and protein expression levels of the EMT markers were measured (FIGS. 58 and 59). As a result, transfection of Axl or Mer siRNA reversed both Gas6 induced E-cadherin increase and α-SMA and N-cadherin decrease. This suggests that Gas6-induced E-cadherin increase and α-SMA and N-cadherin decrease in LA-4 cells result from induction of Axl or Mer expression.

In addition, the present inventors attempted to confirm the effect of Axl or Mer on EMT regulatory transcription factor expression and non-Smad TGF-β1 signaling by Gas6 in LA-4 cells.

LA-4 cells were transfected with Axl or Mer specific siRNA or negative-control siRNA, incubated for 48 hours, and mRNA and protein expression levels of EMT regulatory transcription factors were measured (FIG. 60). As a result, mRNA expressions of Snai1, Zeb1, and Twist1 inhibited by Gas6 were all increased. In addition, both phosphorylation of extracellular signal-regulated kinase (ERK) and Akt inhibited by Gas6 was increased (FIGS. 61 and 62). This suggests that Gas6-induced EMT transcription factor and ERK and Akt phosphorylation inhibition in LA-4 cells are derived from induction of Axl or Mer expression.

Experimental Example 7: Effect of Gas6 on EMT in Primary Mouse AT II Cells

7-1. Effect of Gas6 Pretreatment on TGF-β1-Induced EMT in Primary Mouse AT II Cells

We sought to determine whether Gas6 treatment regulates TGF-β-induced EMT processes in primary mouse AT II cells. After 20 hours of treatment with 400 ng / ml of Gas6 on AT II cells, the cells were treated with 10 ng / ml of TGF-β for 48 hours or 72 hours, and EMT marker mRNA and protein expression levels were confirmed. mRNA expression levels were measured based on Hprt mRNA (FIGS. 63 and 64). The data suggest that biosynthetic mediators synthesized and secreted after Gas6 treatment could block TGF-β signals in autocrine fashion in AT II cells. In addition, the present inventors confirmed that TGF-β treatment after 20 hours of treatment with Gas6 on AT II cells inhibited the mRNA expression of Snai1 / 2, Zeb1 / 2 and Twist1 induced by TGF-β ( 65). The data suggest that the transcriptional regulators are reduced to prevent the EMT process in AT II cells after Gas6 treatment.

7-2. Identification of COX-2-signals for Gas6-induced EMT inhibition in primary mouse AT II cells

We sought to confirm the regulation of COX-2 signaling by Gas6 in primary mouse AT II cells.

We treated 400 ng / ml of Gas6 hourly (0, 1, 2, 3, and 6 hours) to primary mouse AT II cells and measured COX-2 mRNA expression levels (FIG. 66). COX-2 mRNA expression levels showed a maximum after 1 hour of Gas6 treatment and gradually decreased.

Next, to further confirm the functional association of COX-2 signals in the anti-EMT effect of Gas6, we invited NS-398, a highly selective COX-2 inhibitor, for 1 hour prior to treatment with Gas6. Was treated. As a result, NS-398 reversed all of Gas6's effects on E-cadherin loss and synthesis of α-SMA and N-cadherin, and decreased Snai1, Zeb1 and Twist1 mRNA expression at the TGF-β-induced gene level (Fig. 67 and FIG. 68).

Next, the inventors of the present invention, such as EP2 (E-prostanoid-2 receptor) antagonist (AH-6809) or DP2 antagonist (BAY-u3405) in primary mouse AT II cells one hour before TGF-β treatment in the presence or absence of Gas6 Antagonists of PGE ₂ -or PGD ₂ -specific receptors were treated. As a result, the anti-EMT effect of Gas6 was significantly reversed by the antagonists of EP2 and DP2 (FIGS. 69 and 70).

The results suggest that anti-EMT effects in AT II cells are mediated primarily by COX-2 derived PGE ₂ and PGD ₂ signals, respectively, via EP2 and DP2 receptors.

7-3. Confirmation of RhoA Pathway-dependent HGF-signaling for Gas6-induced EMT Inhibition in Primary Mouse AT II Cells

The present inventors attempted to confirm the regulation of RhoA pathway-dependent HGF-signaling by Gas6 in primary mouse AT II cells.

Gas6 treatment in primary mouse AT II cells resulted in a gradual increase in Gas6 induced HGF mRNA expression (FIG. 71).

Next, to inhibit RhoA / Rho kinase signal, primary mouse AT II cells were transfected with RhoA-specific siRNA for 24 hours or treated with Rho kinase inhibitor Y-27632 for 1 hour before Gas6 stimulation. As a result, both RhoA knockdown and Rho kinase inhibition reversed the effect of Gas6 on TGF-β-induced EMT (FIGS. 72 and 73). In addition, the anti-EMT effect of Gas6 was reversed even when c-Met signal was inhibited by treating AT II cells with PHA-665752, a c-Met antagonist, 1 hour before TGF-β treatment (FIGS. 74 and 75).

The data strongly suggest that RhoA / Rho kinase-dependent HGF production in AT II cells also mediates the anti-EMT effect of Gas6 via c-Met signaling by autosecretion.

Experimental Example 8: Effect of Gas6 pretreatment on TGF-β1-induced EMT in human lung adenocarcinoma cells (A549 cells)

The present inventors attempted to determine whether Gas6 administration in A549 cells regulates the TGF-β-induced EMT process.

After 20 hours of treatment with 400 ng / ml of Gas6 in A549 cells, 10 ng / ml of TGF-β was treated for 72 hours, and EMT marker mRNA and protein expression levels were confirmed. mRNA expression levels were determined based on Hprt mRNA (FIGS. 76 and 77). The data suggest that biosynthetic mediators synthesized and secreted after Gas6 treatment could block TGF-β signals in A549 cells in a self-secreting manner.

In addition, the present inventors confirmed that the mRNA expression of Snai1 / 2, Zeb1 / 2, and Twist1 induced by TGF-β was inhibited as a result of TGF-β treatment after Gas6 treatment for 20 hours in A549 cells (FIG. 78). The data suggest that the transcriptional regulators are reduced to prevent the EMT process in A549 cells after Gas6 treatment.

Experimental Example 9: Effect of Gas6 on EMT Inhibition in Animal Model of Pulmonary Fibrosis

9-1. Inhibitory Effects of Gas6 on Bleomycin-Induced EMT in Animal Models of Pulmonary Fibrosis

The present inventors attempted to determine whether Gas6 administration regulates the EMT process in lung epithelial cells in vivo.

C57BL / 6 mice were divided into five groups, respectively, as a control group, a gas6 group (Gas6), a bleomycin group (BLM), and a Gas6 + bleomycin group (Gas6 + BLM). 50 μg / kg of Gas6 was administered for each taxon prior to daily bleomycin administration. Primary mouse AT II cells were isolated from the lungs of mice 14 days after bleomycin administration.

The morphology of the isolated mouse cells was confirmed. As a result, it was confirmed that the cells of the bleomycin-administered mice were converted to spindle-like morphology, but the cells of the mice to which Gas6 was administered before the bleomycin administration decreased the spindle-like morphology (FIG. 79).

In addition, each mRNA level was measured to confirm the expression patterns of EMT markers (E-cadherin, N-cadherin and α-SMA) and EMT-activated transcription factors (Snai1, Zeb1 and Twist1) (FIGS. 80 and 81). . Mice treated with bleomycin showed decreased expression of E-cadherin and increased expression of N-cadherin and α-SMA, but it was confirmed at the mRNA level that the expression of EMT markers was suppressed when Gas6 was treated. In addition, it was confirmed that mRNA expression of Snai1, Zeb1, and Twist1 induced by bleomycin were all inhibited when Gas6 was treated. The results suggest that Gas6 treatment may inhibit the bleomycin-induced EMT in pulmonary fibrosis mouse models.

9-2. COX-2 Derived PGE in an Animal Model of Pulmonary Fibrosis ₂ , PGD ₂  Effect of Gas6 treatment on HGF and HGF secretion

We sought to determine whether Gas6 administration regulates the PGE ₂ , PGD ₂ and HGF pathways in vivo.

C57BL / 6 mice were divided into five groups, respectively, as a control group, a gas6 group (Gas6), a bleomycin group (BLM), and a Gas6 + bleomycin group (Gas6 + BLM). 50 μg / kg of Gas6 was administered for each taxon prior to daily bleomycin administration. Primary mouse AT II cells were isolated from the lungs of mice 14 days after bleomycin administration. MRNA, PGE ₂ , PGD ₂ , HGF and TGF-β production of COX-2 and HGF were confirmed in isolated mouse cells.

As a result, the mRNA level of COX-2 increased more than 2 times compared to the control group due to Gas6 administration, PGE ₂ , which was not secreted from the control group, was secreted more than 4000 pg / ml, and PGD ₂ protein level was about 4 times that of the control group. Increased in degree (FIGS. 82 and 83). The results suggest that Gas6-induced EMT inhibition in the lung fibrosis mouse model results from increased COX-2 expression, PGE ₂ and PGD ₂ production.

In addition, mRNA levels of HGF were increased more than three times compared to the control group due to Gas6 administration, HGF was secreted more than 2500 pg / ml, TGF-β protein level increased by bleomycin was reduced due to Gas6 administration (Fig. 84 And FIG. 85). The results suggest that inhibition of Gas6-induced EM in the pulmonary fibrosis mouse model results from increased HGF and TGF-β inhibition.

9-3. Effect of Gas6 Treatment on Changes of Bleomycin-induced EMT Markers and Collagen Deposition in Animal Models of Pulmonary Fibrosis

We sought to determine whether Gas6 administration in vivo regulates changes in EMT markers and collagen deposition.

C57BL / 6 mice were divided into five groups, respectively, as a control group, a gas6 group (Gas6), a bleomycin group (BLM), and a Gas6 + bleomycin group (Gas6 + BLM). 50 μg / kg of Gas6 was administered for each taxon prior to daily bleomycin administration. Primary mouse AT II cells were isolated from the lungs of mice 14 and 21 days after bleomycin administration. Each isolated mouse cell was western blotted using anti-E-cadherin, anti-N-cadherin, anti-α-SMA, anti-type 1 collagen (collagen 1) or anti-fibronectin antibody. Relative Density of each band was determined based on A-tubulin.

As a result, the levels of E-cadherin reduced by bleomycin in mouse lung tissues at days 14 and 21 were elevated due to Gas6 administration, and N-cadherin, α-SMA, type 1 collagen and fibronectin levels increased by bleomycin. Decreased due to Gas6 administration (FIGS. 86 and 87).

In addition, collagen accumulation in the entire lung was measured for hydroxyproline levels on day 21 (FIG. 88). As a result, the level of hydroxyproline increased by bleomycin decreased to a level similar to that of the control due to Gas6 administration.

The results suggest that fibrosis caused by bleomycin in vivo is ameliorated by Gas6 administration.

Taken together, Gas6 induces the secretion production of PGE ₂ , PGD ₂ and HGF in epithelial cells, and induces TGF-β by acting in a self-secreting manner via signaling pathways through their receptors. It can be seen that it has an anti-EMT effect of inhibiting EMT.

In addition, Gas6 secreted COX-2 and RhoA pathway-dependent HGF based on Axl or Mer signaling pathways, and had the same effect as LA-4 cells in primary mouse AT II cells and human lung adenocarcinoma cells. It can be seen.

In addition, animal experiments confirmed that Gas6 administration had an excellent prophylactic and therapeutic effect on fibrosis by inhibiting EMT induced by bleomycin in vivo.

Therefore, Gas6 can be very useful for the prevention or treatment of fibrosis.

From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

<110> EWHA UNIVERSITY-INDUSTRY COLLABORATION FOUNDATION <120> A composition comprising a Gas6 protein or an receptor activator          about for preventing or treating fibrosis <130> P17-066-REA-EWHA <150> KR 10-2017-0079974 <151> 2017-06-23 <160> 35 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> COX-2 siRNA-sense <400> 1 cuaugauagg agcauguaa 19 <210> 2 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> COX-2 siRNA-antisense <400> 2 uuacaugcuc cuaucauag 19 <210> 3 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> RhoA siRNA-sense <400> 3 gaagucaagc auuucugucu ua 22 <210> 4 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> RhoA siRNA-antisense <400> 4 gacagaaaug cuugacuucu u 21 <210> 5 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Axl siRNA-sense <400> 5 gagauggaca gauccuaga 19 <210> 6 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Axl siRNA-antisense <400> 6 ucuaggaucu guccaucuc 19 <210> 7 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Mer siRNA-sense <400> 7 cacaguuuua uccugauga 19 <210> 8 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Mer siRNA-antisense <400> 8 ucaucaggau aaaacugug 19 <210> 9 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> control siRNA-sense <400> 9 ccuacgccac caauuucgu 19 <210> 10 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> control siRNA-antisense <400> 10 acgaaauugg uggcguagg 19 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> E-cadherin primer-F <400> 11 gcactcttct cctggtcctg 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> E-cadherin primer-R <400> 12 tatgaggctg tgggttcctc 20 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> N-cadherin primer-F <400> 13 cctccagagt ttactgccat gac 23 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> N-cadherin primer-R <400> 14 ccaccactga ttctgtatgc cg 22 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> alpha-SMA primer-F <400> 15 ccaccgcaaa tgcttctaag t 21 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> alpha-SMA primer-R <400> 16 ggcaggaatg atttggaaag g 21 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Snai1 primer-F <400> 17 cccaaggccg tagagctga 19 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Snai1 primer-R <400> 18 gcttttgcca ctgtcctcat c 21 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Snai2 primer-F <400> 19 atcctcacct cgggagcata 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Snai2 primer-R <400> 20 tgccgacgat gtccatacag 20 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Zeb1 primer-F <400> 21 attcagctac tgtgagccct gc 22 <210> 22 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Zeb1 primer-R <400> 22 cattctggtc ctccacagtg ga 22 <210> 23 <211> 22 <212> DNA <213> Artificial Sequence <220> Zeb2 primer-F <400> 23 gcagtgagca tcgaagagta cc 22 <210> 24 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Zeb2 primer-R <400> 24 ggcaaaagca tctggagttc cag 23 <210> 25 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Twist1 primer-F <400> 25 tcgacttcct gtaccaggtc ct 22 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Twist1 primer-R <400> 26 ccatcttgga gtccagctcg 20 <210> 27 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> COX-1 primer-F <400> 27 cgatctggct tcgtgaac 18 <210> 28 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> COX-1 primer-R <400> 28 gagctgcagg aaatagcc 18 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COX-2 primer-F <400> 29 gggagtctgg aacattgtga 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COX-2 primer-R <400> 30 gtgcacattg taagtaggtg 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> H223 primer-F <400> 31 caccccttgg gagtattgtg 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> H223 primer-R <400> 32 gggacatcag tctcattcac 20 <210> 33 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> HPRT primer-F <400> 33 ccagtgtcaa ttatatcttc aac 23 <210> 34 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> HPRT primer-R <400> 34 cagactgaag agctactgta atg 23 <210> 35 <211> 674 <212> PRT <213> Artificial Sequence <220> <223> Gas6 <400> 35 Met Pro Pro Pro Pro Gly Pro Ala Ala Ala Leu Gly Thr Ala Leu Leu   1 5 10 15 Leu Leu Leu Leu Ala Ser Glu Ser Ser His Thr Val Leu Leu Arg Ala              20 25 30 Arg Glu Ala Ala Gln Phe Leu Arg Pro Arg Gln Arg Arg Ala Tyr Gln          35 40 45 Val Phe Glu Glu Ala Lys Gln Gly His Leu Glu Arg Glu Cys Val Glu      50 55 60 Glu Val Cys Ser Lys Glu Glu Ala Arg Glu Val Phe Glu Asn Asp Pro  65 70 75 80 Glu Thr Glu Tyr Phe Tyr Pro Arg Tyr Gln Glu Cys Met Arg Lys Tyr                  85 90 95 Gly Arg Pro Glu Glu Lys Asn Pro Asp Phe Ala Lys Cys Val Gln Asn             100 105 110 Leu Pro Asp Gln Cys Thr Pro Asn Pro Cys Asp Lys Lys Gly Thr His         115 120 125 Ile Cys Gln Asp Leu Met Gly Asn Phe Phe Cys Val Cys Thr Asp Gly     130 135 140 Trp Gly Gly Arg Leu Cys Asp Lys Asp Val Asn Glu Cys Val Gln Lys 145 150 155 160 Asn Gly Gly Cys Ser Gln Val Cys His Asn Lys Pro Gly Ser Phe Gln                 165 170 175 Cys Ala Cys His Ser Gly Phe Ser Leu Ala Ser Asp Gly Gln Thr Cys             180 185 190 Gln Asp Ile Asp Glu Cys Thr Asp Ser Asp Thr Cys Gly Asp Ala Arg         195 200 205 Cys Lys Asn Leu Pro Gly Ser Tyr Ser Cys Leu Cys Asp Glu Gly Tyr     210 215 220 Thr Tyr Ser Ser Lys Glu Lys Thr Cys Gln Asp Val Asp Glu Cys Gln 225 230 235 240 Gln Asp Arg Cys Glu Gln Thr Cys Val Asn Ser Pro Gly Ser Tyr Thr                 245 250 255 Cys His Cys Asp Gly Arg Gly Gly Leu Lys Leu Ser Pro Asp Met Asp             260 265 270 Thr Cys Glu Asp Ile Leu Pro Cys Val Pro Phe Ser Met Ala Lys Ser         275 280 285 Val Lys Ser Leu Tyr Leu Gly Arg Met Phe Ser Gly Thr Pro Val Ile     290 295 300 Arg Leu Arg Phe Lys Arg Leu Gln Pro Thr Arg Leu Leu Ala Glu Phe 305 310 315 320 Asp Phe Arg Thr Phe Asp Pro Glu Gly Val Leu Phe Phe Ala Gly Gly                 325 330 335 Arg Ser Asp Ser Thr Trp Ile Val Leu Gly Leu Arg Ala Gly Arg Leu             340 345 350 Glu Leu Gln Leu Arg Tyr Asn Gly Val Gly Arg Ile Thr Ser Ser Gly         355 360 365 Pro Thr Ile Asn His Gly Met Trp Gln Thr Ile Ser Val Glu Glu Leu     370 375 380 Glu Arg Asn Leu Val Ile Lys Val Asn Lys Asp Ala Val Met Lys Ile 385 390 395 400 Ala Val Ala Gly Glu Leu Phe Gln Leu Glu Arg Gly Leu Tyr His Leu                 405 410 415 Asn Leu Thr Val Gly Gly Ile Pro Phe Lys Glu Ser Glu Leu Val Gln             420 425 430 Pro Ile Asn Pro Arg Leu Asp Gly Cys Met Arg Ser Trp Asn Trp Leu         435 440 445 Asn Gly Glu Asp Ser Ala Ile Gln Glu Thr Val Lys Ala Asn Thr Lys     450 455 460 Met Gln Cys Phe Ser Val Thr Glu Arg Gly Ser Phe Phe Pro Gly Asn 465 470 475 480 Gly Phe Ala Thr Tyr Arg Leu Asn Tyr Thr Arg Thr Ser Leu Asp Val                 485 490 495 Gly Thr Glu Thr Thr Trp Glu Val Lys Val Val Ala Arg Ile Arg Pro             500 505 510 Ala Thr Asp Thr Gly Val Leu Leu Ala Leu Val Gly Asp Asp Asp Val         515 520 525 Val Pro Ile Ser Val Ala Leu Val Asp Tyr His Ser Thr Lys Lys Leu     530 535 540 Lys Lys Gln Leu Val Val Leu Ala Val Glu Asp Val Ala Leu Ala Leu 545 550 555 560 Met Glu Ile Lys Val Cys Asp Ser Gln Glu His Thr Val Thr Val Ser                 565 570 575 Leu Arg Glu Gly Glu Ala Thr Leu Glu Val Asp Gly Thr Lys Gly Gln             580 585 590 Ser Glu Val Ser Thr Ala Gln Leu Gln Glu Arg Leu Asp Thr Leu Lys         595 600 605 Thr His Leu Gln Gly Ser Val His Thr Tyr Val Gly Gly Leu Pro Glu     610 615 620 Val Ser Val Ile Ser Ala Pro Val Thr Ala Phe Tyr Arg Gly Cys Met 625 630 635 640 Thr Leu Glu Val Asn Gly Lys Ile Leu Asp Leu Asp Thr Ala Ser Tyr                 645 650 655 Lys His Ser Asp Ile Thr Ser His Ser Cys Pro Pro Val Glu His Ala             660 665 670 Thro pro        

Claims (8)

A pharmaceutical composition for preventing or treating fibrosis, comprising Gas6 (Growth arrest-specific 6) protein as an active ingredient. delete The method of claim 1, wherein the fibrosis is lung, kidney, liver, heart, brain, blood vessels, joints, intestines, skin, soft tissue, bone marrow, penis, peritoneum, lens, muscles, spine, testes, ovaries, breast, thyroid gland, tympanic membrane , Pancreas, gallbladder, bladder or prostate. The pharmaceutical composition of claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 1, wherein the composition is administered at a dose of 100 μg / kg to 500 μg / kg. The pharmaceutical composition of claim 1, wherein the composition is administered orally or parenterally. The pharmaceutical composition of claim 1, wherein the composition inhibits EMT (epithelial to mesenchymal transition) of cells. An in vitro composition for inhibiting epithelial to mesenchymal transition (EMT) of lung or kidney cells containing a gas arrest (Growth arrest-specific 6) protein.

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