Separate Cis-acting DNA Elements Control Cell Type- and Tissue-specific Expression of Collagen Binding Molecular Chaperone HSP47

Abstract

HSP47 is a collagen-binding heat shock protein and is assumed to act as a molecular chaperone in the biosynthesis and secretion of procollagen. As the synthesis of HSP47 is closely correlated with that of collagen in various cell lines and tissues, we performed a promoter/ reporter assay using HSP47-producing and nonproducing cells. 280 base pairs (bp(s)) of upstream promoter were shown to be necessary for the basal expression but not to be enough for the cell type-specific expression. When the first and the second introns were introduced downstream of this 280-bp region, marked up-regulation of the reporter activity was observed in HSP47-producing cells but not in nonproducing cells. This was confirmed in transgenic mice by staining the lacZ gene product under the control of the 280-bp upstream promoter and the introns. Staining was observed in skin, chondrocytes, precursor of bone, and other HSP47/collagen-producing tissues. A putative Sp1-binding site at ؊210 bp in the promoter, to which Sp3 and an unidentified protein bind, was shown to be responsible for this up-regulation when combined with the introns. However no difference in the binding to this probe was observed between HSP47-producing and nonproducing cells. The responsible region for cell type-specific upregulation was found to be located in a 500-bp segment in the first intron. On electrophoresis mobility shift assay using this 500-bp probe, specific DNA-protein complexes were only observed in HSP47-producing cell extracts. These results suggest that two separate elements are necessary for the cell type-specific expression of the hsp47 gene; one is a putative Sp1-binding site at ؊210 bp necessary for basal expression, and the other is a 500-bp region within the first intron, required for cell typespecific expression.Heat shock proteins (HSPs) 1 are a highly conserved set of proteins that can be induced by heat shock and other environmental stresses. Generally, most stress proteins are expressed not only under stress conditions but also under normal growth conditions and known to play important roles in protein folding and assembly. They are now generally known as molecular chaperones. The mechanism for heat induction of HSPs is well studied and now known to be transcriptionally regulated by the interaction of heat shock factors with heat shock elements (HSEs), which locate in the promoter region of HSPs (1).HSP47 was identified as a collagen-binding 47-kDa glycoprotein (pI ϭ 9.0) (2) and proved to be a heat shock protein (3-6). This protein belongs to the serine protease inhibitor (serpin) superfamily containing a serpin signature sequence (7). HSP47 resides in the endoplasmic reticulum (ER), as inferred from the presence of a carboxyl-terminal RDEL sequence similar to the ER retrieval signal, KDEL (8 -13). HSP47 binds to nascent procollagen chains in the ER of collagen-secreted cells and dissociates from them before reaching the cis-Golgi apparatus (4, 6, 14). Thus HSP47 functions as a collagen-specific molecular chaperone (15).The expression of HS...

Advanced Glycation End Products Increase Collagen-specific Chaperone Protein in Mouse Diabetic Nephropathy

“…Procollagen polypeptides form a complex with HSP47 in the endoplasmic reticulum, which plays an important role as a collagen-specific molecular chaperone in the intracellular processing/folding of procollagen molecules (23,24). The crucial role of HSP47 in regulating biosynthesis of collagen molecules has been reported previously (25), and transcriptional regulation for HSP47 expression was clarified (26,27). However, its role in kidney diseases in relation to sclerosis/ fibrosis in diabetic nephropathy and IgA nephropathy is completely unknown.…”

The Kruppel-like Factor Zf9 and Proteins in the Sp1 Family Regulate the Expression of HSP47, a Collagen-specific Molecular Chaperone

“…The plasmid pact-␤-galactosidase, which bears the chicken ␤-actin promoter upstream of lacZ, was used as an internal control to normalize transfection efficiency. ␤-Galactosidase assays were performed as described previously (15).…”

“…However, the cell type-specific expression of hsp47 could not be attributed to this promoter region alone. Hirata et al (15) also showed that the first two introns, located upstream of the translation initiation site, are necessary in addition to the 280-bp promoter region for the tissuespecific expression of hsp47. Considering these previous results, we made systematic deletion constructs fused to the luciferase gene to identify the cis-acting element(s) in the first and the second introns that are responsible for the constitutive expression of hsp47.…”

“…Activate Expression-In a previous study, Hirata et al (15) reported that a 280-bp region of the promoter upstream of the transcriptional start site is necessary for basal level expression of hsp47 in murine cells. However, the cell type-specific expression of hsp47 could not be attributed to this promoter region alone.…”

“…In a previous paper (15), we demonstrated that both the 280-bp promoter region and the first and second introns are required for the tissue-specific expression of HSP47 in transgenic mice harboring a ␤-galactosidase reporter gene under the control of these elements. In vitro promoter analysis using a luciferase reporter gene revealed that the Sp1 binding site at Ϫ210 bp is necessary for the basal expression of hsp47, and the addition of a downstream intron region caused a marked upregulation of reporter activity in HSP47-and collagen-producing cells but not in nonproducing cells.…”