KR102414380B1 - Pharmaceutical composition for preventing or treating hypertrophic scar comprising CPEB1 or CPEB4 inhibitor - Google Patents

Abstract

Post-burn hypertrophic scar (HTS) is a form of excessive skin fibrosis characterized by skin scarring and is common in patients following burn injury. In addition, more than 50% of HTS is accompanied by inflammation.
Cytoplasmic polyadenylation element binding (CPEB) proteins are major mRNA-binding proteins that control the translation of several mRNAs. However, their potential role in treating skin fibrosis and scarring is unknown. Therefore, in this study, we aimed to investigate the siRNA-mediated knockdown effect of CPEB1 or CPEB4 in human THP-1 macrophages and dermal fibroblasts treated with LPS and TGF-β1. We found that CPEB1 and CPEB4 mRNA and protein levels were significantly increased in LPS-treated THP-1 cells and TGF-β1-treated fibroblasts. CPEB1 and CPEB4 knockdown inhibited the LPS-activated TAK1 signaling cascade by reducing the levels of TNF-α and phosphorylated TAK1, p38, ERK, JNK and NF-κB-p65 in THP-1 cells. CPEB1 and CPEB4 knockdown also attenuated TGF-β1 activated Smad-dependent and Smad-independent signaling cascades by decreasing levels of TAK1, p38, ERK, JNK and phosphorylated Smad 2 and Smad 1/5/8 in fibroblasts . In addition, CPEB1 or CPEB4 knockdown significantly reduced the levels of fibrosis markers including α-SMA, type I collagen and fibronectin in fibroblasts. Our findings indicate that CPEB1 and CPEB4 are involved in the regulation of TAK1 and Smad signaling in human macrophages and dermal fibroblasts. This activity may play an important role in the skin scar response.

Description

Pharmaceutical composition for preventing or treating hypertrophic scar comprising CPEB1 or CPEB4 inhibitor {Pharmaceutical composition for preventing or treating hypertrophic scar comprising CPEB1 or CPEB4 inhibitor}

Post-burn hypertrophic scar (HTS) is a form of excessive skin fibrosis characterized by skin scarring, commonly occurring in patients following burn injury. In addition, more than 50% of hypertrophic scars after burns are accompanied by inflammation.

Cytoplasmic polyadenylation element binding (CPEB) proteins are major mRNA-binding proteins that control the translation of several mRNAs. However, their potential role in treating skin fibrosis and scarring is unknown. Therefore, in the present invention, the present inventors tried to investigate the siRNA-mediated knockdown effect of CPEB1 or CPEB4 in human THP-1 macrophages and skin fibroblasts treated with LPS (Lipopolysaccharide) and TGF-β1. We found that CPEB1 and CPEB4 mRNA and protein levels were significantly increased in LPS-treated THP-1 cells and TGF-β1-treated fibroblasts. CPEB1 and CPEB4 knockdown inhibited the LPS-activated TAK1 signaling cascade by reducing the levels of TNF-α and phosphorylated TAK1, p38, ERK, JNK and NF-κB-p65 in THP-1 cells. CPEB1 and CPEB4 knockdown also attenuated TGF-β1 activated Smad-dependent and Smad-independent signaling cascades by decreasing levels of TAK1, p38, ERK, JNK and phosphorylated Smad 2 and Smad 1/5/8 in fibroblasts . In addition, CPEB1 or CPEB4 knockdown significantly reduced the levels of fibrosis markers including α-SMA, type I collagen and fibronectin in fibroblasts. The results of this study indicate that CPEB1 and CPEB4 are involved in the regulation of TAK1 and Smad signaling in human macrophages and dermal fibroblasts. This activity of CPEB1 and CPEB4 may play an important role in the skin scar response.

Mammalian cytoplasmic polyadenylation element binding (CPEB) proteins, including CPEB1-4, are sequence-specific mRNA-binding proteins that control poly(A) tail elongation and polyadenylation-induced translation [1]. Studies involving frog (Xenopus) oocytes, rodent cells and animal experiments confirmed the biological function of the CPEB protein in healthy and diseased individuals. CPEB1 is involved in insulin signaling and glucose metabolism [2], CPEB4 regulates cell survival and cancer progression [3,4], and CPEB1 and CPEB4 both regulate cell mitosis [5]. CPEB1 and CPEB4 were particularly strongly expressed in the liver of cirrhosis patients, suggesting that abnormal CPEB expression is involved in the pathology of hepatic fibrosis [6].

Lipopolysaccharide (LPS), derived from the outer membrane of most Gram-negative bacteria, is a potent monocyte/macrophage activator that contributes to the inflammatory process [7]. Binding of LPS to Toll-Like Receptor (TLR) 2/4 on the surface of macrophages initiates a series of signaling cascades leading to the production of pro-inflammatory mediators including TNF-α, IL-1β and IL-6 [8,9]. Of these, TGF-β1-activated kinase 1 (TAK1), also known as mitogen-activated protein 3 kinase 7 (MAP3K7), is a molecule downstream of the TLR-mediated activation pathway, and extracellular signal-regulated kinase 1/2 (ERK1/2) , functions as intracellular signaling hubs regulating classical inflammatory pathways, including c-Jun N-terminal kinase (JNK) and NF-κB signaling [9,10].

Hypertrophic scars after burns refer to scars that protrude upwards during the healing process after burns. The incidence of hypertrophic scars after burns is very high, accounting for 60% to 90% of burn patients. The physical and mental pain and economic damage of burn patients is a very large and serious social problem. Hypertrophic scars start to appear around 1 to 2 weeks as the burn wound heals, and most of them grow for 4 to 6 months, up to 2 years, and are accompanied by various symptoms. and joint contractures. In addition, unlike normal skin, it does not include skin appendages such as sweat glands, fat glands, hair, and hair follicles. Hypertrophic scars after burns are not only unsightly, but also cause various sensory abnormalities such as pain and itching, and cause serious functional impairment depending on the area.

Hypertrophic scarring after burns is a very common and serious problem that occurs in burn patients, but there is still no clear pathogenesis and no established effective treatment methods. There is an urgent need to identify pathophysiological mechanisms of development and develop disease-specific therapeutics based on the mechanism.

Hypertrophic scar (HTS), characterized by excessive extracellular matrix (ECM) deposition, is a fibroproliferative skin scar that often occurs after thermal or traumatic injury in deep skin tissue [11]. Mechanical stress is an important factor in hypertrophic scar development and induces the transcription of collagen in vitro and induces the development of hypertrophic scar features in vivo, such as scarring and epidermal thickening, mast cell invasion, angiogenesis and hypercellular proliferation. In addition, mechanical stress stimulates the differentiation of fibroblasts into myofibroblasts during burn wounds in humans [12,13]. Importantly, mechanical stimulation activates latent TGF-β1 via αvβ6 integrin-mediated stimulation of the intracellular small GTPase Rho pathway [14]. Prolonged or severe inflammation is another important cause of hypertrophic scar formation in burn patients [14,15]. Therefore, hypertrophic scars are considered inflammatory pathological lesions. Indeed, the inflammatory cytokines IL-6 and IL-1β are overexpressed in hypertrophic scar tissue fibroblasts [15,16]. Moreover, macrophage polarization to the M2 phenotype induces hypertrophic scar formation and excessive TGF-β1 secretion, while ensuring normal wound healing to the M1 phenotype [17,18]. TGF-β1 is an important molecular factor in hypertrophic scar formation because it acts as a potent inducer of fibroblast proliferation and differentiation, leading to excessive ECM synthesis [11]. This pathological role of TGF-β1 in tissue fibrosis involves Smad-dependent and Smad-independent signaling pathways [11]. TAK1, an important upstream signaling molecule in the Smad-independent signaling pathway activated by TGF-β1, is involved in inflammatory or fibrotic diseases of various organs such as kidney, lung and heart [19-21].

In this study, we investigated the effect of siRNA-mediated CPEB1 or CPEB4 knockdown on LPS-induced proinflammatory and TGF-β1-induced profibrotic responses in THP-1 macrophages and dermal fibroblasts, respectively. We found that CPEB1 or CPEB4 knockdown inhibits LPS-induced pro-inflammatory signaling that occurs through TAK1, P38, JNK and ERK kinases and that NF-κB in THP-1 cells and TGF-β1-induced Smad-induced Smad- in dermal fibroblasts. We found that it significantly inhibited NF-kB in both fibrotic-dependent and Smad-independent fibrillation signaling pathways. Thus, CPEB1 and CPEB4 and pathways regulating their expression may affect cellular pathways involved in skin fibrosis.

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An object of the present invention is to provide a more effective preventive or therapeutic agent for hypertrophic scar formation.

In the present invention, we showed that both CPEB1 and CPEB4 were induced by LPS and TGF-β1 in THP-1 macrophages and dermal fibroblasts, respectively. These cells are an important source of extracellular matrix (ECM) in fibrotic scars. Furthermore, we demonstrated that siRNA-mediated CPEB1 or CPEB4 knockdown inhibited LPS-activated inflammatory signaling in THP-1 cells and TGF-β1-induced fibrosis signaling in fibroblasts. These findings suggest that CPEB1 and CPEB4 may play important roles in fibrotic scar formation through activation of inflammatory and fibrotic signaling.

Hypertrophic scarring is an important concern during the rehabilitation of patients with severe burns. It not only limits the patient's eventual recovery, but also complicates and worsens treatment outcomes. Hypertrophic scars usually occur at the site of injury and damage the appearance, and when hypertrophic scars are formed in the joints, dysfunction may occur [11]. Although the exact mechanism of hypertrophic scar formation is still not well understood, it is widely accepted that a number of factors are involved, including mechanical stress, inflammatory cells, cytokines, chemokines, and growth factors. Macrophages play an important role in hypertrophic scar formation during wound healing. Classically activated M1 macrophages produce pro-inflammatory cytokines (IL-6 and TNF-α), whereas alternatively activated M2 macrophages secrete trophic factors (PDGF and TGF-β1) [23] . IL-6 stimulates ECM synthesis in fibroblasts and inhibits MMP-1 and MMP-3, which have anti-scarring effects because they degrade ECM [24]. PDGF and TGF-β1 actively induce myofibroblast formation and stimulate excessive collagen synthesis in these myofibroblasts [11,25]. Immunohistochemical analysis showed an increase in macrophage numbers in a human hypertrophic scar-like nude mouse model. However, CXCR4 antagonist treatment reduced the number of macrophages, TGF-β1 expression, myofibroblast differentiation and fibrotic gene expression [26,27].

In our preliminary study, qPCR analysis in 20 pairs of hypertrophic scar tissue and normal skin tissues obtained from patients showed that the mRNA levels of CPEB1 and CPEB4 in hypertrophic scar tissues were 1.8 of those of CPEB1 and CPEB4 in normal skin tissues, respectively. fold and 2.2 fold (data not shown). These results suggest that CPEB1 and CPEB4 are involved in hypertrophic scar formation.

Knockdown of all CPEB proteins (CPEB1-4) has been shown to increase TAK1 and MAPK signaling, prolong IκBα degradation, activate NF-κB, and increase inflammatory cytokine production in LPS-treated mouse RAW 264.7 macrophages [28]. These results indicate that CPEB protein controls TAK1 expression and subsequently mediates the inflammatory response. Nevertheless, it has been proposed to investigate the role of individual subtypes in the inflammatory response. However, the results showed that siRNA-mediated CPEB1 or CPEB4 knockdown reduced LPS-induced phosphorus protein expression of TAK1, p38, JNK, ERK1/2, IκBα and p65 in human monocyte-derived THP-1 macrophages (Fig. 1A). Knockdown also reduced mRNA and protein levels of TNF-α (Fig. 2B, C).

Previously, the expression of TNF-α in hypertrophic scar tissue was lower than that in normal trophic scar tissue and normal skin tissue [29]. This unexpectedly low TNF-α expression may be associated with reduced apoptosis in hypertrophic scars [23]. A low TNF-α concentration promotes the proliferation of fibroblasts and produces excessive ECM, which contributes to hypertrophic scar formation [30]. Therefore, mechanisms regulating TNF-α synthesis may be involved in the fibrotic scar formation process. In the present invention, CPEB1 or CPEB4 knockdown reduced TNF-α production in LPS-treated THP-1 cells.

Targeting TGF-β-mediated Smad-dependent signaling has been considered as a novel therapeutic strategy for fibrosis [31]. However, recent studies have revealed a pathological role of TAK1 signaling in fibrosis. TAK1 signaling is a TGF-β1-activated Smad-independent pathway [21], and its downstream targets p38, JNK, ERK, and NF-κB are involved in the pathogenic profibrotic response [32,33]. Burn patients were found to have significantly higher serum TGF-β levels than control patients [34]. Furthermore, we previously showed that the expression of TGF-β1, α-SMA, type I collagen and fibronectin in fibroblasts derived from hypertrophic scar tissue was increased compared to normal skin-derived fibroblasts [35]. In the present invention, CPEB1 or CPEB4 knockdown inhibited TGF-β1-mediated Smad-dependent and Smad-independent signaling (Fig. 4A, B) and reduced TGF-β1-induced upregulation of α-SMA, type I collagen and fibronectin. (Figure 4C and D). To determine whether TAK1 signaling regulates Smad-dependent signaling in fibroblasts, we performed inhibitor studies using (5Z)-7-oxoseenol, a potent TAK1 inhibitor [36]. We found that 30 μM (5Z)-7-oxoseenol treatment reduced phosphorylation of SMAD2 and SMAD1/5/8 and phosphorylation of TAK1-downstream signaling molecules JNK and ERK, but not p38. revealed (data not shown). These results suggest that CPEB1 and CPEB4 knockdown partially inhibits the Smad-dependent signaling pathway through TAK1.

The present invention provides a pharmaceutical composition for preventing or treating hypertrophic scarring, comprising an expression inhibitor or an activity inhibitor for CPEB1 or CPEB4.

In addition, the present invention provides that the CPEB1 or CPEB4 expression inhibitor is any one selected from the group consisting of antisense nucleotides complementary to CPEB1 or CPEB4 gene mRNA, shRNA (short hairpin RNA) and siRNA (short interfering RNA) It provides a composition, characterized in that.

As the siRNA for CPEB1 or CPEB4, a product commercially available from Sigma-Aldrich, etc. can be used, and the siRNA for CPEB1 or CPEB4 is based on the CPEB1 or CPEB4 gene sequence. Those of ordinary skill in the art to which the present invention belongs The siRNA sequence generation program widely known to those of ordinary skill in the art to which the present invention pertains can be used to easily generate and prepare a sequence. In addition, it is apparent to those skilled in the art that siRNA for CPEB1 or CPEB4 gene can inhibit expression of CPEB1 or CPEB4 by binding to CPEB1 or CPEB4 gene.

In addition, as the shRNA for CPEB1 or CPEB4, a product commercially available from Sigma-Aldrich, etc. can be used. Alternatively, the shRNA for CPEB1 or CPEB4 is commonly used in the art based on the CPEB1 or CPEB4 gene sequence. Those with the knowledge of the present invention can easily generate and prepare a sequence using a well-known shRNA sequence generation program to those of ordinary skill in the art to which the present invention pertains. In addition, it is clear to those skilled in the art that shRNA for CPEB1 or CPEB4 gene can inhibit the expression of CPEB1 or CPEB4 by binding to CPEB1 or CPEB4 gene.

In addition, the present invention provides a composition characterized in that the CPEB1 or CPEB4 protein activity inhibitor is an antibody that binds to CPEB1 or CPEB4 protein.

As the antibody binding to the CPEB1 or CPEB4 protein, commercially available products such as Sigma-Aldrich may be used.

The composition for preventing or treating hypertrophic scarring comprising the CPEB1 or CPEB4 expression inhibitor or activity inhibitor of the present invention as an active ingredient contains 0.0001 to 50% by weight of the active ingredient based on the total weight of the composition.

The composition of the present invention may contain at least one active ingredient that exhibits the same or similar function to the CPEB1 or CPEB4 protein expression inhibitor or activity inhibitor. The composition of the present invention may be administered orally or parenterally during clinical administration, and may be used in the form of general pharmaceutical formulations.

The composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration. A pharmaceutically acceptable carrier may be used in a mixture of saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and one or more of these components, and, if necessary, an antioxidant , buffers, bacteriostatic agents, and other conventional additives may be added. In addition, diluents, dispersing agents, surfactants, binders and lubricants may be additionally added to form injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets, and may act specifically on target organs. A target organ-specific antibody or other ligand may be used in combination with the carrier. Furthermore, it can be preferably formulated according to each component by an appropriate method in the art or by using a method disclosed in Remington's Pharmaceutical Science (Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA).

The composition of the present invention may include a lyophilized composition, particularly for the composition of an isotonic sterile solution or an injectable solution upon addition of sterile water or appropriate physiological saline. The dosage of the active ingredient used varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of disease, etc., and the daily dosage is about 0.0001 to 100 mg /kg, preferably 0.001 to 10 mg/kg, may be administered once to several times a day divided.

The composition of the present invention can be used alone or in combination with chemotherapy, a method using a biological response modifier, and other therapeutic agents.

The pharmaceutical composition for preventing or treating hypertrophic scarring, comprising an inhibitor of CPEB1 or CPEB4 protein activity or expression inhibitor of the present invention, inhibits Smad-dependent signaling pathway through TAK1, and TGF-β1-induced fibrosis signaling in fibroblasts By suppressing the hypertrophic scar formation can be suppressed.

Figure 1. LPS treatment increased CPEB1 and CPEB4 expression in THP-1 cells. (A, B) RT-PCR analysis results 24 hours after siRNA transfection. Results are expressed as fold change for levels in negative control siRNA treated cells. (C, D) Western blotting results 48 hours after siRNA transfection. For quantitative analysis, protein levels were normalized to those of GAPDH.
All samples were analyzed in triplicate, and experiments were performed independently in triplicate. Data are expressed as mean ± standard deviation. *p < 0.01 compared to negative control siRNA-transfected cells; †p < 0.01 compared to negative control siRNA-transfected cells and LPS-treated cells. si-Ct, negative control siRNA; si-CP1, CPEB1 siRNA; si-CP4, CPEB4 siRNA.
Figure 2. CPEB1 or CPEB4 knockdown inhibited LPS-induced inflammatory signaling in THP-1 cells. (A) Western blotting results after LPS treatment for 0.5, 1 or 4 hours. CPEB1 and CPEB4 knockdown reduced LPS-induced phosphorylation levels of TAK1, p38, JNK, ERK, IκBα and NF-κB-p65. (B) RT-qPCR analysis results after LPS treatment for 4 h. CPEB1 and CPEB4 knockdown reduced LPS-induced increase in TNF-α mRNA level. These results are expressed as fold change relative to the level of negative control siRNA-treated cells. (C) ELISA results for TNF-α levels after LPS treatment for 4 hours. CPEB1 and CPEB4 knockdown reduced LPS-induced increase in TNF-α protein levels.
All samples were analyzed in triplicate, and experiments were performed independently in triplicate. Data are presented as mean ± standard deviation. * p < 0.01 compared to negative control siRNA-transfected cells; † p < 0.01 compared to negative control siRNA-transfected cells and LPS-treated cells. si-Ct, negative control siRNA; si-CP1, CPEB1 siRNA; si-CP4, CPEB4 siRNA.
Figure 3. TGF-β1 treatment increased CPEB1 and CPEB4 expression in fibroblasts. (A, B) RT-qPCR analysis results 24 hours after siRNA transfection. Results are expressed as fold change relative to the level of negative control siRNA-treated cells. (C, D) Western blotting results 48 hours after siRNA transfection. For quantitative analysis, protein levels were normalized to GAPDH levels.
All samples were analyzed in triplicate, and experiments were performed independently in triplicate. Data are presented as mean ± standard deviation. * p < 0.01 compared to negative control siRNA-transfected cells; † p < 0.01 compared to negative control siRNA-transfected cells and LPS-treated cells. si-Ct, negative control siRNA; si-CP1, CPEB1 siRNA; si-CP4, CPEB4 siRNA.
Figure 4. CPEB1 or CPEB4 knockdown inhibited fibrosis signaling by TGF-β1 in fibroblasts (A, B). Western blotting results after TGF-β1 treatment for 0.5, 1 or 4 hours. CPEB1 and CPEB4 knockdown reduced TGF-β1-induced phosphorylation of TAK1, p38, JNK and ERK involved in Smad-independent signaling and TGF-β1-induced induction of SMAD2 and SMAD1/5/8 involved in Smad-dependent signaling phosphorylation was attenuated. (C) RT-qPCR analysis results after TGF-β1 treatment for 24 h. CPEB1 and CPEB4 knockdown reduced TGF-β1-induced increases in α-SMA, type I collagen and fibronectin mRNA levels. Results are expressed as fold change compared to the level of negative control siRNA-treated cells. (D) Western blotting results after TGF-β1 treatment for 48 hours. CPEB1 and CPEB4 knockdown reduced TGF-β1-induced α-SMA, type I collagen and fibronectin protein levels increase.
All samples were analyzed in triplicate, and experiments were performed independently in triplicate. Data are presented as mean ± standard deviation. * p < 0.01 compared to negative control siRNA-transfected cells and TGF-β1-treated cells. si-Ct, negative control siRNA; si-CP1, CPEB1 siRNA; si-CP4, CPEB4 siRNA; COL-I, type I collagen; FN, fibronectin.
Figure 5. Effect of CPEB1 and CPEB4 siRNAs on expression of different subtypes. (A, B) RT-PCR analysis at 24 h after siRNA transfection. Results are expressed as fold change in relation to the level of negative control siRNA treated cells. All samples were analyzed in triplicate, and experiments were performed independently in triplicate. Data are presented as mean ± standard deviation. * p <0.01 compared to cells transfected with negative control siRNA. si-Ct, negative control siRNA; si-CP1, CPEB1 siRNA; si-CP4, CPEB4 siRNA; CP2, CPEB2; CP4, CPEB4.
Figure 6. Effect of CPEB1 and CPEB4 knockdown on cell proliferation. (A, B) THP-1 and fibroblast proliferation results at 24 and 48 hours after siRNA transfection. Results are expressed as fold change in relation to the level of negative control siRNA treated cells. All samples were analyzed in triplicate, and experiments were performed independently in triplicate. Data are presented as mean ± standard deviation. * p <0.05 compared to negative control siRNA-transfected cells. si-Ct, negative control siRNA; si-CP1, CPEB1 siRNA; si-CP4, CPEB4 siRNA.
Figure 7. CPEB1 and CPEB4 knockdown inhibited TGF-β1-induced fibroblast migration. (A) Results of fibroblast migration in an in vitro wound healing assay. Images were taken under an optical microscope (scale bar, 1 mm). (B) Quantitative analysis of fibroblast migration. All samples were analyzed in triplicate, and experiments were performed independently in triplicate. Data are presented as mean ± standard deviation. * p <0.01 is the value compared to TGF-β1-treated cells. si-Ct, negative control siRNA; si-CP1, CPEB1 siRNA; si-CP4, CPEB4 siRNA.
Figure 8. Quantitative analysis of p-TAK1, p-p38, p-JNK, p-ERK, p-IKBα and p-p65. The intensity of each band was measured and the phosphorylated form was normalized to the intensity of the total form. All experiments were performed independently in triplicate. Data are presented as mean ± standard deviation. * p <0.01 compared to LPS-treated cells. si-Ct, negative control siRNA; si-CP1, CPEB1 siRNA; si-CP4, CPEB4 siRNA.
Figure 9. CPEB1 and CPEB4 knockdown inhibited TGF-β receptor I expression in fibroblasts. (A, B) Results of Western blotting after TGF-β1 treatment for 0.5, 1 and 4 h. CPEB1 knockdown reduced TGF-β1-induced TGF-β receptor I expression. TGF-β receptor II expression was not altered. All experiments were performed independently in triplicate. Data are presented as mean ± standard deviation. * p <0.01 is the value compared to TGF-β1-treated cells. si-Ct, negative control siRNA; si-CP1, CPEB1 siRNA; si-CP4, CPEB4 siRNA.
Figure 10. Quantitative analysis of p-TAK1, p-p38, p-JNK and p-ERK. The intensity of each band was measured and the phosphorylated form was normalized to the intensity of the total form. All experiments were performed independently in triplicate. Data are presented as mean ± standard deviation. * p <0.01 is the value compared to TGF-β1-treated cells. si-Ct, negative control siRNA; si-CP1, CPEB1 siRNA; si-CP4, CPEB4 siRNA.
Figure 11. Quantitative analysis of p-SMAD2 and p-SMA1/5/8. The intensity of each band was measured and the phosphorylated form was normalized to the intensity of the total form. All experiments were performed independently in triplicate. Data are presented as mean ± standard deviation. * p < 0.01 compared to TGF-β1-treated cells. si-Ct, negative control siRNA; si-CP1, CPEB1 siRNA; si-CP4, CPEB4 siRNA.

Hereinafter, the configuration of the present invention will be described in more detail with reference to specific embodiments. However, it is apparent to those skilled in the art that the scope of the present invention is not limited only to the description of the examples.

1. Cell Culture

THP-1 cells purchased from the Korean Cell Line Bank (KCLB, Seoul Korea) were treated with 10% fetal bovine serum (FBS; Biowest, Riverside, MO, USA) and an antibiotic-antibacterial agent (Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with RPMI 1640 (containing stable glutamine and 25 mM HEPES) at 1 × 10 ⁶ /ml and cultured in tissue culture dishes for 2 days. Cells were differentiated using 10 ^-8 M phorbol-12-myristate-13-acetate (PMA) for 48 hours. Human primary dermal fibroblasts (passage 2) were purchased from PromoCell (Heidelberg, Germany) and obtained from Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS (in all experiments, fibroblasts passages 4-6 were used); Biowest, MO, USA) were cultured.

2. siRNA transfection and LPS and TGF - β1 process

Negative controls, CPEB1 and CPEB4 SMARTpool siRNAs were purchased from Dharmacon (Lafayette, CO, USA). Both differentiated THP-1 cells and fibroblasts were grown to 50-70%, washed using Dulbecco's buffered saline (Biowest), and cultured in antibiotic-free medium for 24 hours. Each siRNA (50 nM) was transfected for 5 h using the StemFect RNA transfection kit (Stemgent, Cambridge, MA, USA) according to the manufacturer's instructions. The medium was then replaced with supplemental medium. Then, the cells were cultured in a medium containing 0.1% FBS for 6 hours and treated with LPS or TGF-β1. For the analysis of inflammatory signals, THP-1 cells were treated with LPS (1 μg/ml) for 0.5, 1 or 4 hours, and for the analysis of TNF-α concentration in the culture medium, THP-1 cells were treated with LPS (1 μg/ml). ml) for 4 hours. To evaluate TGF-β1 signaling in fibroblasts, fibroblasts were treated with TGF-β1 (5 ng/ml) for 0.5, 1 or 4 h. To evaluate α-smooth muscle actin (α-SMA), collagen-I and fibronectin mRNA and protein levels, fibroblasts were treated with TGF-β1 for 24 or 48 hours.

3. Enzyme-Linked Immunosorbent Assay (ELISA)

To determine the TNF-α concentration in the cell culture medium, the supernatant was collected from LPS-treated THP-1 cells. The concentration of TNF-α was measured using ELISA (PeproTech, Rocky Hill, NJ, USA) according to the manufacturer's instructions.

4. reverse transcription Quantitative real-time polymerase chain reaction (RT- qPCR )

Total RNA from THP-1 cells or fibroblasts was extracted using the ReliaPrep™ RNA Miniprep system (Promega, Madison, WI, USA) according to the manufacturer's instructions. cDNA was synthesized using 500 ng RNA and PrimeScript™ RT master mix (Takara, Siga, Japan). Real-time PCR was performed on a LightCycler 96 system (Roche, Basel, Switzerland) using a PCR premix (Takara), 50 ng cDNA and 0.5 μM primers. The primers were: CPEB1, 5'-GGTGTCCTCCCAAAGGTAATA-3' (forward) and 5'-CAAGCCTGAGCAAGGATCG-3'(reverse); CPEB2, 5'-CGATATTATGAGAGCAGAG-3' (forward) and 5'-CTATTGGAAAGAGGGAAG-3'(reverse); CPEB3, 5'-GCTGATATAATGTGGAGGAA-3' (forward) and 5'-CCAGGAAGGCATTGTTAA-3'(reverse); CPEB4, 5'-TGGGGATCAGCCTCTTCATA-3' (forward) and 5'-CAATCCGCCTACAAACACCT-3'(reverse); IL-6, 5'-TGCTTGTTCCTC AGCCTT-3' (forward) and 5'-CAGAGGGCTGATTAGAGAGAGGT-3'(reverse); α-SMA, 5′-CCGACCGAATGCAGAAGGA-3′ (forward) and 5′-ACAGAGTATTTGCGCTCCGAA-3′ (reverse); COLA1A1, 5'-ATGTTCAGCTTTGTGGACCTC-3' (forward) and 5'-CTGTACGCAGGTGATTGGTG-3'(reverse); and fibronectin, 5'-CCAGTCCACAGCTATTCCTG-3' (forward) and 5'-ACAACCACGGATGAGCTG-3' (reverse). Estimated mRNA levels were normalized to GAPDH mRNA levels using the 2 ^{- ΔΔCt} method [22].

5. western blotting

THP-1 cells or fibroblasts were lysed in cold RIPA buffer (Biosesang, Seongnam, Korea) containing a protease inhibitor (Sigma, St. Louis, MO, USA) and a phosphatase inhibitor (Roche). Samples (30 μg protein/well) were digested by 8% SDS-PAGE and electrotransferred to PVDF membranes (Merck Millipore, Billerica, MA, USA). The following primary antibodies were used: rabbit polyclonal anti-CPEB1 and anti-CPEB4 (Thermo Fisher Scientific, Waltham, MA, USA); Rabbit polyclonal anti-phospho-TAK1, anti-TAK1, anti-p38, anti-phospho-JNK, anti-JNK, anti-phospho-ERK and anti-β-actin-rabbit monoclonal anti-phospho-SMAD2 and anti -SMAD1—and mouse polyclonal anti-phospho-p38 and anti-ERK (Cell Signaling Technology Danvers, MA, US); rabbit polyclonal anti-NF-κB-p65 and anti-collagen-I, mouse polyclonal anti-α-SMA, and mouse monoclonal anti-fibronectin and anti-SMAD2 (Abcam, Cambridge, UK); mouse monoclonal anti-phospho-IκBα, anti-IκBα and anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit polyclonal anti-phospho-SMAD1/5/8 (Merck Millipore); rabbit polyclonal TGF β receptor I antibody (1:500) and rabbit polyclonal TGF β receptor II antibody (1:1000) (Abcam, Cambridge, UK). Peroxidase-conjugated anti-mouse or anti-rabbit IgG (1 : 3,000, Merck Millipore) was used as secondary antibody. Images were acquired using a chemiluminescence imaging system (ATTO, Tokyo, Japan), and band density was analyzed using CS Analyzer 4 software (ATTO).

6. Cell proliferation assay

Cell proliferation was assessed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay Kit (Promega, Madison, WI, USA). THP-1 (1 x 10 ⁵ cells/well) and fibroblasts (2 x 10 ⁴ /well) were grown in 96 well plates prior to siRNA transfection. After replacement with 100 μl of culture medium, CellTiter 96®; 20 μl of AQueous One Solution reagent was added. Plates were incubated in a cell culture incubator at 37°C or for 2 hours, and absorbance was measured at 490 nm using a 96-well plate reader (BioTek, Winooski, VT, USA). Proliferation was calculated as follows: Proliferation (%) = (Sample Absorbance-Background Absorbance)/(Control Sample Absorbance-Background Absorbance) Х 100.

7. Cell Migration Assay

Fibroblast migration assays were performed as per reference (39). Briefly, fibroblasts (2 x 10 ⁴ /well) were grown on inserts (Ibidi, GmbH, Planegg, Germany), and after 24 h of siRNA transfection, the inserts were removed and the cells were separated from the cell-free niche (defined as wounds) began to move. Cells were imaged at 24 and 48 h under a light microscope (IX 70, Olympus, Tokyo, Japan), and the number of cells migrating to the gap was analyzed with Image J software (NIH, Bethesda, MD, USA). TGF-β1 treated cells as a positive control. Quantitative analysis of migration assays was expressed as a percentage of negative control siRNA treated cells.

8. Statistical Analysis

All results are presented as mean ± standard deviation. The Mann-Whitney U test was used for comparison between the two groups. Statistical analysis was performed using PASW Statistical 24 (SPSS Inc., Chicago, IL, USA). p < 0.01 was considered to indicate significance.

Result 1. CPEB1 with siRNA CPEB4 siRNA Cross-reactivity measurements between

RT PCR was performed to confirm the specificity of CPEB1 and CPEB4 siRNAs in THP-1 cells and fibroblasts. No significant cross-reactivity of CPEB1 siRNA in CPEB4 expression or CPEB4 siRNA in CPEB1 expression was observed with THP-1. In addition, CPEB1 siRNA and CPEB4 slightly increased CPEB2 mRNA levels by 135% and 128%, respectively ( FIG. 5 ). CPEB1 siRNA increased CPEB4 mRNA level by 320% compared to the level in fibroblasts transfected with negative control siRNA, but no cross-reactivity of CPEB4 siRNA to CPEB1 expression was observed. Both CPEB1 siRNA and CPEB4 siRNA increased CPEB2 mRNA levels in fibroblasts by 225% and 328%, respectively ( FIG. 5B ). Neither CPEB1 siRNA nor CPEB4 siRNA affected CPEB3 mRNA levels in THP-1 cells or fibroblasts.

Result 2. THP -1 in cells and fibroblasts siRNA Effect of transfection on proliferation

Proliferation of THP-1 and fibroblasts was investigated 24 or 48 hours after CPEB1 siRNA or CPEB4 siRNA transfection using the CellTiter 96® AQueous One Solution Cell Proliferation Assay Kit. CPEB1 or CPEB4 knockdown for 24 h did not affect the proliferation of THP-1 cells. However, after 48 hours of knockdown, THP-1 proliferation was significantly reduced ( FIG. 6A ). In contrast, CPEB1 or CPEB4 knockdown increased the proliferation of fibroblasts after 24 or 48 hours ( FIG. 6B ).

Result 3. For the migration of fibroblasts siRNA Effect of transfection

Fibroblast migration was measured 24 or 48 hours after CPEB1 or CPEB4 siRNA transfection using an in vitro wound healing assay. CPEB1 and CPEB4 knockdown after siRNA transfection significantly reduced fibroblast migration after siRNA transfection compared to TGF-β1 treated cells ( FIG. 7 ).

Result 4. LPS THP -1 in cells CPEB1 and CPEB4 expression was induced.

LPS is a potent stimulator of macrophage-mediated inflammation. In this study, we observed that LPS stimulation significantly increased the mRNA and protein levels of CPEB1 and CPEB4 in THP-1 cells (Fig. 1). However, siRNA-mediated transient knockdown of CPEB1 or CPEB4 suppressed the effect of LPS, and mRNA levels were lower than that of negative control siRNA-transfected cells. Indeed, CPEB1 and CPEB4 siRNA reduced LPS-induced CPEB1 and CPEB4 mRNA levels by 90% and 85% and protein levels by 75% and 70%, respectively, in THP-1 cells ( FIG. 1 ). These results not only confirm the high efficiency of CPEB1 and CPEB4 siRNA transfection, but also suggest that CPEB1 and CPEB4 are involved in the LPS-mediated inflammatory response in THP-1 cells.

Result 5. CPEB1 and CPEB4 knock down is THP -1 in cells LPS -Induced inflammatory signals were suppressed.

To determine whether CPEB1 or CPEB4 modulates LPS-mediated TAK1 signaling, we investigated changes in the TAK1 signaling pathway in LPS-treated CPEB1- or CPEB4-knockdown THP-1 cells using Western blotting. . Compared to untreated controls, cells treated with LPS for 0.5, 1 or 4 h showed significantly increased phosphorylation of TAK1, p38, JNK, ERK, IκBα and NF-κB-p65 ( FIGS. 2A and 8 ). In addition, CPEB1 or CPEB4 knockdown inhibited LPS-activated phosphorylation of these proteins. However, the total levels of these proteins were not significantly different (Fig. 2A, Fig. 8). In addition, RT-qPCR and ELISA revealed that CPEB1 or CPEB4 knockdown reduced TNF-α mRNA (Fig. 2B) and protein (Fig. 2C) levels in THP-1 cells and culture medium, respectively. However, both mRNA and protein levels of TNF-α in CPEB1- or CPEB4-knockdown cells were higher than in negative control siRNA-transfected cells ( FIGS. 2B and C). These results indicate that LPS-activated inflammatory signals are regulated by CPEB1 and CPEB4 expression.

Results 6. In fibroblasts TGF - β1 is CPEB1 and CPEB4 induce expression.

TGF-β1 is widely used to develop an in vitro fibrosis model. Therefore, we investigated the effect of TGF-β1 treatment on CPEB1 and CPEB4 expression in fibroblasts. TGF-β1 treatment for 24 h was observed to significantly increase both mRNA and protein expression of CPEB1 and CPEB4 in fibroblasts ( FIG. 3 ). However, CPEB1 and CPEB4 siRNA transfections reduced TGF-β1-induced mRNA expression of CPEB1 and CPEB4 by 80% and 88%, respectively, and protein expression of CPEB1 and CPEB4 by 60% and 73%, respectively. In addition, these levels were lower than those of fibroblasts transfected with negative control siRNA ( FIG. 3 ). These results confirm the siRNA transfection efficiency and suggest the involvement of CPEB1 and CPEB4 in TGF-β1-mediated fibrosis signaling in fibroblasts.

Result 7. CPEB1 and CPEB4 Knockdown in fibroblasts TGF - β1 - induced Smad -independent and Smad-independent signaling pathways were inhibited.

The Smad-dependent signaling pathway and the TAK1 signaling pathway, a TGF1-β1-induced Smad-independent signaling pathway, together contribute to tissue fibrotic scar formation [19]. Therefore, to study whether CPEB1 or CPEB4 knockdown altered the TGF-β1 signaling pathway, we first detected changes in the protein levels of TGF-β receptor I (TbRI) and TGF-β receptor II (TbR II) in fibroblasts. Western blotting was performed for this purpose. TGF-β1-induced protein levels of TbRI and TbR II were significantly reduced by CPEB1 knockdown. However, CPEB4 knockdown did not affect TGF-β1-induced protein levels of TbRI and TbR II ( FIG. 9 ). Next, we measured the Smad-independent signaling pathway. CPEB1 and CPEB4 knockdown significantly reduced the levels of phosphorylated TAK1, p38, JNK and ERK in fibroblasts treated with TGF-β1 for 0.5, 1 or 4 h ( FIGS. 4A and 10 ). Finally, we investigated changes in SMAD2 and SMAD1/5/8, components of Smad-dependent signaling. TGF-β1 treatment for 0.5, 1 or 4 h resulted in a marked increase in phosphorylation of SMAD2 and SMAD1/5/8 (Fig. 4B, Fig. 11), whereas CPEB1 or CPEB4 knockdown caused TGF-β1-induced SMAD2 and SMAD1/ 5/8 phosphorylation was significantly inhibited ( FIGS. 4B and 11 ). These results indicate that CPEB1 and CPEB4 regulate TGF-β1-induced fibrosis signaling. To further explore the inhibitory effect of CPEB1 and CPEB4 knockdown on fibrotic scar formation, the expression of several molecular markers of skin fibrosis or scarring was evaluated. RT-qPCR and Western blotting revealed reduced mRNA and protein levels of α-SMA, type I collagen and fibronectin in TGF-β1-treated CPEB1- or CPEB4-knockdown fibroblasts, respectively ( FIGS. 4C and D ). These results indicate that CPEB1 and CPEB4 may play a role in fibrotic scar formation by regulating Smad-dependent and Smad-independent signaling pathways.

Claims (10)

CPEB1 (Cytoplasmic polyadenylation element binding protein 1) mRNA expression inhibitor comprising siRNA (short interfering RNA) or shRNA (short hairpin RNA) for the CPEB1 gene, a pharmaceutical composition for preventing or treating hypertrophic scarring.
A CPEB4 (Cytoplasmic polyadenylation element binding protein 4) mRNA expression inhibitor, comprising siRNA (short interfering RNA) or shRNA (short hairpin RNA) for the CPEB4 gene, a pharmaceutical composition for preventing or treating hypertrophic scarring. delete delete delete delete delete delete delete delete

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