JP3688883B2 - Expression regulation region of stress protein HSP47 and use thereof - Google Patents

Description

配列番号：２
配列の長さ：１６５９
配列の型：核酸
鎖の数：２本鎖
トポロジー：直鎖状
配列の種類：ＤＮＡ
由来：マウスゲノム
配列

配列番号：３
配列の長さ：４２７０
配列の型：核酸
鎖の数：２本鎖
トポロジー：直鎖状
配列の種類：ＤＮＡ
由来：マウスゲノム
配列

【図面の簡単な説明】
【図１】図１は、ストレス蛋白質ＨＳＰ４７のマウスゲノムの遺伝子地図を示すものである。
【図２】図２は、ストレス蛋白質ＨＳＰ４７の第１イントロンの上流５．５Ｋｂをプロモーターとした場合のルシフェラーゼアッセイの結果を示すものである。
【図３】図３は、ストレス蛋白質ＨＳＰ４７の転写開始点から上流２８０ｂｐ及び第１イントロン領域を含むプロモーターを用いた場合のルシフェラーゼアッセイの結果を示すものである。
【図４】図４は、ストレス蛋白質ＨＳＰ４７の第１イントロン領域の一部の塩基配列を欠損させたプロモーターを用いた場合のルシフェラーゼアッセイの結果を示すものである。
【図５】図５は、第１エクソン領域の上流２８０ｂｐまでのプロモーターを組み込んだ発現ベクターｐＧＬ２−Ｂａｓｉｃを示す。
【図６】図６は、第１エクソン領域の上流２８０ｂｐ及び第１イントロン領域までのプロモーターを組み込んだ発現ベクターｐＧＬ２−Ｂａｓｉｃを示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a promoter capable of controlling the expression of stress protein HSP47 in which 93 bp in the region from the transcription start point to upstream -280 bp and the first intron in the genomic DNA of stress protein HSP47 is deleted, and its base sequence Alternatively, the present invention relates to a polynucleotide having a base sequence capable of hybridizing thereto. The promoter of the present invention can control the transcription activity of the stress protein HSP47 and can simultaneously control the expression of co-expressed collagen. Accordingly, the present invention relates to control of transcriptional activity of stress protein HSP47 for treating various diseases caused by accumulation of collagen such as cirrhosis, and a pharmaceutical composition comprising these polynucleotides or their complementary strands.
[0002]
[Prior art]
The stress protein HSP47 localized in the endoplasmic reticulum is a collagen-specific molecular chaperone that has specificity for collagen and helps processing and secretion of procollagen in cells.
[0003]
As a result of cDNA cloning, HSP47 was found to be a protein belonging to the serpin family, which is a family of serine-type protease inhibitors, from the amino acid sequence thereof. Normally, serpin family proteins are secreted and function extracellularly as protease inhibitors, but HSP47 has an endoplasmic reticulum retention signal consisting of RDEL (Arg-Asp-Glu-Leu) at the C-terminus. For this reason, HSP47 is localized in the endoplasmic reticulum.
[0004]
On the other hand, a protein localized in the endoplasmic reticulum is known to have a KDEL (Lys-Asp-Glu-Leu) sequence at its C-terminus, and the sequence is trapped by the KDEL receptor present in the cis-Golgi. , Come back on the retrograde transport system from the Golgi to the endoplasmic reticulum, that is, by reciprocating between the endoplasmic reticulum and the Golgi like a shuttle, it is thought that it is localized in the endoplasmic reticulum as a result Has been.
[0006]
HSP47 is considered to have an RDEL sequence instead of its KDEL sequence. In fact, when the RDEL sequence was excised from the C-terminus of HSP47 by genetic engineering, such a mutant HSP47 could no longer remain in the endoplasmic reticulum and was secreted extracellularly.
[0007]
It was confirmed that HSP47 binds to procollagen during synthesis. This is because when a polysome fraction is first taken and immunoprecipitated with an anti-HSP47 antibody, nascent procollagens of various lengths co-precipitate. Secondly, when cells labeled with [ ³⁵ S] methionine for only a very short time were immunoprecipitated with anti-HSP47 antibody and electrophoresed under non-reduction, a sedimentation band was observed at the position of single-chain procollagen. It was revealed from.
[0008]
These indicate that HSP47 is bound to the single-chain procollagen being synthesized, whereas HSP47 can also bind to the three-chain procollagen. This is because in the latter experiment, a band co-precipitated not only at the single-stranded band but also at the triple-stranded position is detected.
[0009]
That is, the stress protein HSP47 binds at the same time as the α chain of procollagen is inserted into the endoplasmic reticulum while synthesizing, and even after two α1 chains and one α2 chain form three chains, It is thought to be bound to three-chain procollagen.
[0010]
Triple chain formation in the endoplasmic reticulum of procollagen begins at the C-terminus. Only after the synthesis to the C-terminus is complete, triple chain formation occurs. In other words, until the polypeptide is synthesized up to the C-terminal, the α chain synthesized so far cannot be folded or aggregated with itself or other α chains. It is. It seems to have some mechanism to prevent it. HSP47 is involved as a molecular chaperone in such α-chain folding / aggregation inhibition.
[0011]
HSP47 is transported from the endoplasmic reticulum to the cis-Golgi while binding to the three-chain procollagen, where it dissociates from the procollagen, and HSP47 itself binds to the KDEL receptor and is transported back to the endoplasmic reticulum. Is sequentially passed through the Golgi apparatus and secreted to the cell surface.
[0012]
In addition to the above-described functions related to the processing of procollagen under normal conditions, HSP47 also has a mechanism for quality control of collagen under stress as a stress protein. That is, when an abnormality occurs in procollagen due to heat stress or the like, a mechanism for inhibiting secretion is provided in order to prevent such abnormal collagen from accumulating in the extracellular matrix. This is a so-called quality control mechanism.
[0013]
By the way, as a substance that inhibits the formation of triple chains of collagen, there is a reagent called α, α′-dipyridyl. α, α'-dipyridyl is an iron chelator, which inhibits hydroxylation of lysine and proline on procollagen and prevents O-glycosylation, thereby suppressing the formation of procollagen triple chains. It is done. Treat cells with this reagent, then pulse-label them with radioactive methionine, chase them for various times, and then immunoprecipitate the cell extract with anti-HSP47 and anti-collagen antibodies. Collagen is almost all secreted extracellularly after 60 minutes, but in the cells treated with α, α'-dipyridyl, almost all procollagen remains in the cell even after 2 hours. confirmed.
[0014]
At this time, when the amount of procollagen immunoprecipitated with anti-HSP47 antibody and coprecipitated with HSP47 was examined, all of the normal cells that were dissociated from HSP47 after 60 minutes were α, α'-dipyridyl. The treated cells have been shown to remain bound to HSP47 after 2 hours.
This is because HSP47 continues to bind to the abnormal procollagen that can no longer form a triple chain, preventing it from being secreted outside the cell, that is, responsible for the quality control mechanism. Because.
[0015]
Furthermore, HSP47 can be thought of as a molecular chaperone with specificity for collagen as described above. On the other hand, a remarkable feature of HSP47 is that it not only has a functionally close relationship with collagen, but also its expression itself has a close correlation with collagen.
That is, a large amount of HSP47 is synthesized in cells that synthesize a large amount of collagen, and conversely, HSP47 is not synthesized at all in cells that do not synthesize collagen.
[0016]
For example, when collagen synthesis is reduced due to malignant transformation (canceration) of cells, HSP47 is suppressed to the same extent. Further, when F9 cells, which are mouse teratocarcinoma cells, are induced to differentiate with retinoic acid or the like, remarkable induction of type IV collagen and the like is observed, but at that time, HSP47 is also dramatically induced.
In addition to such functional aspects, molecular chaberones are correlated with their substrates in expression (Nagata, “Protein Nucleic Acid Enzyme”, Vol. 41, No. 7, 888, 1996).
From the co-expression of HSP47 and collagen as described above, when collagen synthesis was abnormally promoted in a pathological state, it was thought that expression of HSP47 could also be promoted simultaneously. To confirm this, rats were administered carbon tetrachloride and an experimental cirrhosis model was created.
[0017]
Rats are dosed with carbon tetrachloride and livers are collected from week 2 to week 12. When the liver section is stained with Azan, the collagen fibers are stained blue, but in the normal liver, no collagen staining is seen except for the blood vessel wall. When carbon tetrachloride is given, the deposition of collagen fibers begins to be observed from about the 4th week, and at the 12th week, apparent foliar formation is observed, resulting in symptoms of cirrhosis.
RNA was extracted from such liver, and the mRNA levels of HSP47 and collagen were measured by Northern blot analysis. In normal rats, HSP47 mRNA, type I collagen, and type III collagen mRNA were hardly detected. On the other hand, when carbon tetrachloride was administered, the accumulation of collagen mRNA was increased, and it was observed that HSP47 mRNA was also significantly accumulated. Both were already significantly induced from the second week of administration. The degree of progression of liver cirrhosis is evaluated in five stages from pathological specimens, and the correlation between the progression of such pathology and the induction of mRNA for HSP47 and collagen shows a very high correlation coefficient, which correlates with the pathology. It was shown that this is a phenomenon.
[0018]
To examine whether HSP47 is produced in liver parenchymal cells or in other cells present in liver tissue, in situ hybridization using liver samples and antisense to HSP47 and type I collagen (In situ hybridization). As a result, it was confirmed that cells expressing collagen mRNA also expressed HSP47 mRNA. Since it is known that it is Ito cells that produce collagen, in situ hybridization and immunostaining are performed on the same cells using an antibody against desmin, a marker of Ito cells. On the other hand, it was found that it was Ito cells that produced HSP47 mRNA (Nagata, Environmental Science Research Institute Annual Report, Vol. 15, p. 13, 1996).
[0019]
Thus, stress protein HSP47 has a close relationship with lesions accompanied by fibrosis in the body, such as cirrhosis and renal fibrosis, particularly from a clinical point of view. It is synthesized rapidly. Fibrosis such as cirrhosis currently has no means of treatment other than methods such as liver transplantation. If collagen synthesis and accumulation can be suppressed by some method, great progress is expected in the treatment of these diseases.
[0020]
[Problems to be solved by the invention]
The present invention is intended to clarify the transcriptional regulatory (activation) region of HSP47 and to control the synthesis of HSP47 based on this region, and to be useful for the treatment of fibrosis. Specifically, by using this transcription activation region, it is possible to search for a transcription factor that binds to it. Furthermore, the present invention provides a therapeutic method and a therapeutic agent for pathological conditions caused by collagen accumulation including fibrosis by regulating the interaction between a transcription factor and the transcription activation region.
[0021]
[Means for Solving the Problems]
The present invention relates to a transcriptional activation region of stress protein HSP47, which consists of a region comprising 280 bp upstream from the transcription start point and about 100 bp in the first intron in the genomic DNA of stress protein HSP47. By controlling the transcriptional activity of the stress protein HSP47, the expression of co-expressed collagen can be controlled simultaneously. Therefore, the present invention relates to the control of transcriptional activity of stress protein HSP47 for treating various diseases caused by collagen accumulation such as cirrhosis.
[0022]
The present invention relates to a nucleotide sequence in the first intron of genomic DNA of stress protein HSP47, the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing or a nucleotide sequence obtained by deleting, adding, or replacing one or more bases, Alternatively, the present invention relates to a polynucleotide that can hybridize to the base sequence, particularly DNA.
The present invention also relates to a pharmaceutical composition comprising these polynucleotides or their complementary strands, and particularly to a pharmaceutical composition that is a prophylactic / therapeutic agent for diseases caused by collagen accumulation. A preferred pharmaceutical composition of the present invention includes a preventive / therapeutic agent for cirrhosis.
Furthermore, the present invention relates to a promoter capable of controlling the expression of the protein obtained by deleting the base sequence represented by SEQ ID NO: 1 in the first intron in the genomic DNA of the stress protein HSP47, and further from the transcription start site. The present invention relates to the above-mentioned promoter which is deficient in 280 bp upstream.
The present invention also relates to a method for controlling the transcriptional activity of stress protein HSP47 by deleting the nucleotide sequence represented by SEQ ID NO: 1 in the first intron in the genomic DNA of stress protein HSP47, and further, the transcription initiation site The present invention relates to a method for controlling the transcriptional activity of stress protein HSP47 in which 280 bp upstream is deleted.
[0023]
First, since the expression control of HSP47 is at the transcription level, the present inventor performed a promoter analysis for the purpose of identifying a cis element on DNA involved in co-expression with collagen.
All genes of HSP47 were cloned from a mouse genomic library (FIG. 1). The gene encoding HSP47 was composed of six exons of I, II, III, IV, V and VI, and the translation initiation codon was found to be in exon III.
[0024]
The base sequence of the promoter portion about 1.7 kb upstream from the first exon is shown in SEQ ID NO: 2 in the sequence listing. As will be described later, it was found that about 280 bp (CCCGCGGAA ... AGTGAGCTG) from position 1373 (SacII) of this sequence is an important sequence as a promoter.
The base sequence from the first exon region to the third exon region is shown in SEQ ID NO: 3 in the sequence listing. The region up to about 200 bp is the first exon region, the second exon region is near 1250 bp, and the third exon region is from about 3600 bp. A 93 bp base sequence (see SEQ ID NO: 1) of the first intron region described later is a sequence from 836 to 928 bp of the polynucleotide represented by SEQ ID NO: 3.
[0025]
Then, the promoter region from the mouse genomic library was cloned from the transcription start point to 5.5 Kb upstream, and which base sequence of this promoter region was involved in the transcriptional regulation of HSP47 was examined by luciferase assay. . Assuming that the co-expression mechanism is functioning in cells producing collagen and that the transcriptional activity of the base will be high, luciferase in the upstream region of the promoter is performed using the luciferase gene as a reporter. This assay was performed using mouse fibroblast Balb / c3T3 producing collagen.
[0026]
As a result, it was found that having a region from the transcription start point to 280 bp upstream is sufficient for basic transcriptional activity (FIG. 2). Extending the promoter region up to 5.5 Kb upstream did not change the transcriptional activity of the base.
However, this region alone could not support co-expression with collagen, and there was no change in activity in collagen-producing cells or non-collagen-producing cells.
[0027]
Next, a similar luciferase assay was performed in the promoter region containing the downstream intron. That is, when the region containing the first intron was connected downstream of this 280 bp, the transcriptional activity 4 to 6 times as much as that in the promoter region containing the intron was observed in the promoter region containing the intron. In addition, when a similar experiment was performed on human 293 cells that did not produce collagen, the transcriptional activity of the base did not change with or without introns.
Thus, it was found that the region of (280 bp + first intron) supported co-expression with collagen (see FIG. 3). That is, it was found that a cis element involved in co-expression with collagen exists in the intron region of the HSP47 gene.
[0028]
Since the first intron is as long as about 3 kb, it was investigated which region was important by making various deletion mutations. As a result, it was found that the fragment between BstXI-SfiI of the first intron was important. This portion was further examined, and finally, it was found that the transcriptional activation ability of HSP47 was not observed in the two deletion mutations BS14 and BS18 shown in FIG. That is, it became clear that these two regions are important for the co-expression of collagen together with the upstream 280 bp (FIG. 4). When about 100 bp (see SEQ ID NO: 1) including BSI4 and BS18 was connected to the upstream 280 bp promoter (BS21), it was found that it was necessary and sufficient to activate HSP47 transcription alone.
[0029]
A map of the recombinant vector used in these experiments is shown in FIGS. FIG. 5 shows a gene map (Luc280) in which a promoter composed of -280 bp upstream of the transcription start point is incorporated into the expression vector pGL2-Basic. FIG. 6 shows a gene map (Luc280 (III)) in which an expression vector pGL2-Basic is incorporated with a promoter comprising -280 bp upstream of the transcription start point and the first intron region.
[0030]
That is, it was found that the expression of collagen in collagen-producing cells can be controlled by controlling the activity of the BS-14 region or BS-18 region in FIG. Therefore, various diseases caused by the accumulation of collagen such as cirrhosis are prevented by inhibiting the production of the BS-14 region or the BS-18 region of the cells at the diseased site, thereby suppressing the collagen production at the diseased site and performing preventive treatment of the disease. It becomes possible.
As an agent for preventing or treating a disease caused by collagen accumulation of the present invention, an antisense polynucleotide of a polynucleotide encoding the BS-14 region or BS-18 region of FIG. 4 can also be used.
[0031]
The nucleotide sequence of the polynucleotide of the present invention is shown in the sequence listing. “H” used as a base symbol in the sequence listing indicates A, C or T, “K” indicates G or T, “M” indicates A or C, “N” indicates A, C, G or T, “R” represents A or G, “S” represents C or G, “V” represents A, C or G, “W” represents A or T, “Y” represents C or T.
[0032]
【Example】
EXAMPLES Next, although an Example demonstrates this invention in detail, this invention is not limited to these Examples.
[0033]
Example 1 (Cloning of mouse genomic DNA)
Using a mouse HSP47 cDNA (F2) as a probe, a genomic library prepared from mouse liver (purchased from Clontech) was screened by the colony hybridization method. As a result, a 1.2 kb genomic clone was obtained. Using this as a probe, screening was carried out to catch the full length of the gene, and 11 clones were initially obtained. Of these, 2 were the same, so 10 independent clones were obtained. .
Finally, clone 4, which was the longest and encoded the full length of HSP47, could be obtained. Clone 4 was a gene consisting of about 15 kb. For this gene, a restriction enzyme map (see FIG. 1) was prepared, and the gene sequence was determined by a DNA sequencer by an ordinary method. Based on the past data, exon and intron structures were determined based on the obtained cDNA sequence, and finally a genome map as shown in FIG. 1 was prepared.
[0034]
Example 2 (Luciferase assay using upstream region)
The luciferase assay was performed by connecting the firefly luciferase gene as a reporter downstream of 280 bp upstream of the transcription start point of the HSP47 gene (including 38 bp downstream from the transcription start point of the first exon), and cloning site of pGL2control purchased from Clontech Inserted into. As a control, pGL2control (Clontech) having a large T antigen promoter of SV40 virus was used (indicated by “GL2-control” in the figure). In order to correct the introduction efficiency between the target groups, β Using a galactosidase gene as a reporter, a gene linked downstream of the β-actin promoter was simultaneously introduced into the cells.
For introduction into cells, 70,000 cells are implanted in a culture dish having a diameter of 35 mm, and the promoter gene to be examined the next day and the β-galactosidase gene for correction are simultaneously introduced into the cells by the calcium phosphate method. After 4 hours, the culture solution was changed, and after 48 hours of culture, the cells were collected and broken by freeze-thawing. Then, using a certain amount (10 μl) of cell extract, luciferase activity measurement, β-galactosidase activity Was done. Correction was performed by keeping β-galactosidase activity constant. For the activity measurement of luciferase, 100 ul of Picker Gene (manufactured by Toyo Ink Co., Ltd.) was mixed as a substrate, and immediately after mixing, luminescence was measured with a luminometer to determine the activity. In the measurement of β-galactosidase activity, ONPG (o-nitrophenyl b-D-galactoside) was used as a substrate, mixed with the cell extract, incubated at 37 ° C. for 30 minutes, and then the absorbance at 420 nm was measured to determine the activity.
When investigating the promoter activity including the first intron, the entire first intron (1.5 kb) up to the 280 bp upstream of the transcription start point and before the translation start codon is connected, and the luciferase gene is downstream. The one that was connected was used.
[0035]
Example 3 (Luciferase assay with first intron)
Mouse Balb cells and human 293 cells are introduced into the cells by the same method as in Example 2, and the enzyme activity is measured in the same manner. The results are shown in FIG.
[0036]
Example 4 (Luciferase assay in which part of the first intron is deleted)
Mouse Balb cells and human 293 cells are introduced into the cells by the same method as in Example 2, and the enzyme activity is measured in the same manner. The results are shown in FIG.
[0037]
Example 5
The expression vector shown in FIG. 5 was obtained by cloning 5.5 kb of SacI / EagI into pBlueScript, then cleaving it with SacI, and then partially cleaving with a low concentration of AraI, from −5.5 kb (SacI) to +38 kb ( AraI) was obtained. This was inserted into the SacI / XhoI site of pGL2-basic to prepare LUC5.5. Cut with SacI and SacII and end-equilibrium. LUC280 (FIG. 5) was produced.
[0038]
Example 6
In the expression vector shown in FIG. 6, the SalI site was introduced by PCR immediately before the translation start point to produce a fragment of −5.5 kb (SalI) to +3583 kb (SolII), and the nucleotide sequence was confirmed. This was inserted into the SacI / SolI site of GL2-basic to prepare LUC5.5 (III). Cleaved with XhoI and SacII, and subjected to terminal equilibration treatment to produce LUC280 (III) (FIG. 6).
[0039]
【The invention's effect】
The present invention provides a polynucleotide region capable of controlling the transcriptional activity of the stress protein HSP47 that is co-expressed with collagen, thereby effectively preventing and treating various diseases caused by collagen accumulation such as cirrhosis. It becomes possible.
[0040]
[Sequence Listing]
SEQ ID NO: 1
Sequence length: 93
Sequence type: Number of nucleic acid strands: Double-stranded topology: Linear sequence type: DNA
Origin: Mouse genome sequence:

SEQ ID NO: 2
Sequence length: 1659
Sequence type: Number of nucleic acid strands: Double-stranded topology: Linear sequence type: DNA
Origin: Mouse genome sequence

SEQ ID NO: 3
Sequence length: 4270
Sequence type: Number of nucleic acid strands: Double-stranded topology: Linear sequence type: DNA
Origin: Mouse genome sequence

[Brief description of the drawings]
FIG. 1 shows a genetic map of the mouse genome of stress protein HSP47.
FIG. 2 shows the results of a luciferase assay using 5.5 Kb upstream of the first intron of the stress protein HSP47 as a promoter.
FIG. 3 shows the results of a luciferase assay when using a promoter containing 280 bp upstream and the first intron region from the transcription start point of the stress protein HSP47.
FIG. 4 shows the results of a luciferase assay using a promoter in which a partial base sequence of the first intron region of stress protein HSP47 is deleted.
FIG. 5 shows an expression vector pGL2-Basic incorporating a promoter up to 280 bp upstream of the first exon region.
FIG. 6 shows an expression vector pGL2-Basic incorporating a promoter up to 280 bp upstream of the first exon region and up to the first intron region.

Claims (3)

The following polynucleotide (a) or (b):
(A) a polynucleotide comprising the base sequence represented by SEQ ID NO: 1
(B) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 1 and enhances the transcription activity of a promoter that controls transcription of the stress protein HSP47 gene nucleotide. The polynucleotide according to claim 1, wherein the promoter that controls transcription of the stress protein HSP47 gene is a promoter existing from the transcription start point of the stress protein HSP47 gene to 280 base pairs upstream. The polynucleotide according to claim 1 or 2 , wherein the polynucleotide is DNA.

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