Abstract. Heat shock protein 47 (HSP47), a collagenspecific stress protein, has been postulated to be a collagen-specific molecular chaperone localized in the ER. We previously demonstrated that HSP47 transiently associated with newly synthesized procollagen in the ER (Nakai, A., M. Satoh, K. Hirayoshi, and K. Nagata.1992. J. Cell Biol. 117:903-914). In the present work, we examined the location where HSP47 binds to and dissociates from newly synthesized procollagen within the cells, and whether HSP47 associates with nascent single procollagen polypeptide chains and/or with mature triple-helix procollagen. This was accomplished by biochemical coprecipitation with anti-HSP47 and anticollagen antibodies, combined with pulse-label and chase experiments in the presence or absence of various inhibitors for protein secretion, as well as by confocal laser microscopic observation of the cells double stained with both antibodies. We further examined whether the RDEL (Arg-Asp-Glu-Leu) sequence at the COOH terminus of HSP47 can act as an ER-retention signal, as the KDEL sequence does.When the secretion of procollagen was inhibited by the presence of e~, ed-dipyridyl, an iron chelator that inhibits procollagen triple-helix formation, or by the presence of brefeldin A, which inhibits protein transport between the ER and the Golgi apparatus, procollagen was found to be bound to HSP47 during the chase period in the intermediate compartment. In contrast, the dissociation of procollagen chains from HSP47 was not inhibited when procollagen secretion was inhibited by monensin or bafilomycin A1, both of which are known to be inhibitors of post-cis-Golgi transport. These findings suggest that HSP47 and procollagen dissociated between the post-ER and the cis-Golgi compartments.HSP47 was shown to bind to nascent, single-polypeptide chains of newly synthesized procollagen, as well as to the mature triple-helix form of procollagen. HSP47 with the RDEL sequence deleted was secreted out of the cells, which suggests that the RDEL sequence actually acts as an ER-retention signal, as the KDEL sequence does. This secreted HSP47 did not acquire endogycosidase H resistance. The biological significance of the interaction between HSP47 and procollagen in the central secretory pathway, as well as possible mechanisms for this pathway, will be discussed.

HSP47 is a collagen-binding stress protein and is assumed to act as a collagen-specific molecular chaperone during the biosynthesis and secretion of procollagen in the living cell. The synthesis of HSP47 has been reported to correlate with that of collagen in several cell lines.We examined the expression of HSP47 mRNA during the progression of carbon tetrachloride (CC14)-induced liver fibrosis in rats. Northern blot analysis revealed that the expression of HSP47 mRNA was markedly induced during the progression of fibrosis in parallel with al (I) and al (III) collagen mRNAs. By in situ hybridization, the distribution of HSP47 transcripts was similar to that of al (I) collagen and was observed only in cells lining collagen fibrils. These collagen-positive cells were confirmed to be Ito cells by immunohistochemistry for desmin. The absence of high levels of HSP47 mRNA in the liver of rats treated with only a

Abstract. The 47,000-D collagen-binding glycoprotein, heat shock protein 47 (HSP47), is a stress-inducible protein localized in the ER of collagen-secreting ceils. The location and collagen-binding activity of this protein led to speculation that HSP47 might participate in collagen processing. Chemical cmsslinking studies were used to test this hypothesis both before and after the perturbation of procollagen processing.The association of procoLlagen with HSP47 was demonstrated using cleavable bifunctional crosslinking reagents. HSP47 and procollagen were shown to be coprecipitated by the treatment of intact cells with anti-HSP47 or with anticollagen antibodies. Furthermore, several proteins residing in the ER were noted to be crosslinked to and coprecipitated with HSP47, suggesting that these ER-r~ident proteins may form a large complex in the ER. When cells were heat shocked, or when stable triple-helix formation was inhibited by treatment with o~,c~'-dipyridyl, coprecipitation of procollagen with HSP47 was increased. This increase was due to the inhibition of procollagen secretion and to the accumulation of procollagen in the ER. Pulse label and chase experiments revealed that coprecipitated procollagen was detectable as long as procollagen was present in the endoplasmic reticulum of ot,~'-dipyridyltreated cells. Under normal growth conditions, coprecipitated procollagen was observed to decrease after a chase period of 10-15 rain, whereas total procollagen decreased only after 20-25 min. In addition, the intracellular association between HSP47 and procollagen was shown to be disrupted by a change in physiological pH, suggesting that the dissociation of procoUagen from HSP47 is pH dependent. These findings support a specific role for HSP47 in the intracellular processing of procollagen, and provide evidence of a new category of "molecular chaperones" in terms of its substrate specificity and the dissociation mechanism.M EMBRANE proteinS as well as secretory and lysosoreal proteins enter the ER where they are targeted for the secretory pathway. The ER membrane and the membrane enclosed lumen contain many resident proteins that are involved in the processing of secretory proteins. Among these are certain stress proteins. Glucoseregulated protein 78 (GRP78 ~ or immunoglobulin-binding protein [BiP]), a member of the heat shock protein 70 (HSP70) family of proteins, acts like an ATP-dependent intracellular detergent (for review see Pelham, 1989;Rothman, 1989). It associates with nascent proteins to facilitate protein folding and assembly, or with misfolded proteins to prevent secretion of these proteins (Haas and Wabl, 1983; Akira Nakai's present address is

Transcriptional activation of human heat shock protein (HSP) genes by heat shock or other stresses is regulated by the activation of a heat shock factor (HSF). Activated HSF posttranslationally acquires DNA-binding ability. We previously reported that quercetin and some other flavonoids inhibited the induction of HSPs in HeLa and COLO 320DM cells, derived from a human colon cancer, at the level of mRNA accumulation. In this study, we examined the effects of quercetin on the induction of HSP70 promoterregulated chloramphenicol acetyltransferase (CAT) activity and on the binding of HSF to the heat shock element (HSE) by a gel mobility shift assay with extracts of COLO 320DM cells. Quercetin inhibited heat-induced CAT activity in COS-7 and COLO 320DM cells which were transfected with plasmids bearing the CAT gene under the control of the promoter region of the human HSP70 gene. Treatment with quercetin inhibited the binding of HSF to the HSE in whole-cell extracts activated in vivo by heat shock and in cytoplasmic extracts activated in vitro by elevated temperature or by urea. The binding of HSF activated in vitro by Nonidet P-40 was not suppressed by the addition of quercetin. The formation of the HSF-HSE complex was not inhibited when quercetin was added only during the binding reaction of HSF to the HSE after in vitro heat activation. Quercetin thus interacts with HSF and inhibits the induction of HSPs after heat shock through inhibition of HSF activation.Physiologic stress, including heat shock, enhances the synthesis of a limited number of intracellular proteins, the so-called heat shock proteins (HSPs) (19). The heat shock response has been observed in all cells so far tested, and some of the HSPs have been well conserved throughout evolution. In higher organisms, the induction of HSPs by heat shock or other stresses is regulated at the transcriptional and translational levels. The transcription of heat shock genes is regulated by the cis-acting heat shock element (HSE) in the promoter region and the trans-acting heat shock factor (HSF). The HSE consensus sequence was defined as the repeat of a 5-bp unit, NGAAN or NTTCN (2,28), where N is any nucleotide, and the molecular cloning of HSF from yeast and Drosophila cells has been reported (5,36,38). In Saccharomyces cerevisiae, HSF is already bound to the HSE under normal conditions, and transcriptional activation is induced after heat shock at least partly through the phosphorylation of HSF, whereas in Drosophila and mammalian cells, HSF acquires DNA-binding ability only after heat shock through posttranslational modification of HSF (18, 34).We have reported that quercetin and several other flavonoids inhibit the synthesis of HSPs, including HSP110, HSP90, HSP70, HSP47, HSP40, and HSP28, induced by heat shock, azetidine, or sodium arzenite treatment in two human cancer cell lines, HeLa and COLO 320DM cells (12). Quercetin inhibited the induction of HSP70 at the level of mRNA accumulation (12).Flavonoids are a group of dyes commonly contained in higher plants (...

Keywords: flavonoids/heat shock proteins/human cancer cell lines ABSTRACT. Cells exposed to several forms of stress, such as heat shock, transiently synthesize a group of proteins called heat shock proteins (hsps). Although many stressors other than heat shock are known to induce hsps, inhibitors of hsp expression have never been reported. Here we show that quercetiri and several other flavonoids inhibit the synthesis of hsps induced by heat shock in two human cell lines, Hela cells and COLO320 DMcells. Quercetin inhibited the induction of hsp70 at the level of mRNAaccumulation. This is the first report to describe the inhibition of hsp expression by reagents.Whencells or organisms are exposed to heat shock,

Chromatin structure can modulate gene expression by limiting transcription factor access to gene promoters. We examined sequence elements of the Drosophila hsp70 promoter for their ability to facilitate the binding of the transcription factor, heat shock factor (HSF), to chromatin. We assayed HSF binding to various transgenic heat shock promoters in situ by measuring amounts of fluorescence at transgenic loci of polytene chromosomes that were stained with an HSF antibody. We found three promoter sequences that influence the access of HSF to its binding sites: the GAGA element, sequences surrounding the transcription start site, and a region in the leader of hsp70 where RNA polymerase I1 arrests during early elongation. The GAGA element has been shown previously to disrupt nucleosome structure. Because the two other critical regions include sequences that are required for stable binding of TFIID in vitro, we examined the in vivo occupancy of the TATA elements in the transgenic promoters. We found that TATA occupancy correlated with HSF binding for some promoters. However, in all cases HSF accessibility correlated with the presence of paused RNA polymerase 11. We propose that a complex promoter architecture is established by multiple interdependent factors, including GAGA factor, TFIID, and RNA polymerase 11, and that this structure is critical for HSF binding in vivo.

A 47-kDa heat-shock protein (HSP47) is a major collagen-binding stress protein residing in the endoplasmic reticulum, and is assumed to be a molecular chaperone specific to collagen. Twodimensional gel electrophoresis and immunoprecipitation studies showed that the expression of HSP47 was significantly induced during the differentiation of mouse teratocarcinoma F9 cells by treatment with retinoic acid alone or with retinoic acid and dibutyryladenosine 3',5'-phosphate. The induction of type-IV collagen was also observed during F9-cell differentiation. For further analysis, we cloned cDNA encoding mouse HSP47 from a cDNA library of BALB/c 3T3 cells and performed Northern-blot analysis. The cDNA contained a signal sequence at the N-terminus and an endoplasmic-reticulum-retention signal, RDEL, at the C-terminus. An homology search revealed that mouse HSP47, as well as chick HSP47, belonged to the serine protease inhibitor superfamily. While chick HSP47 mRNA was 4.5 kb with a long (2-kb) 3' untranslated region, mouse and human HSP47 mRNA were 2.5 kb, with a 0.8-kb 3' untranslated region. Northern-blot analysis revealed that the concurrent induction of HSP47 and type-IV collagen during F9-cell differentiation, and the transient induction of HSP47 after heat shock was regulated at the level of mRNA accumulation. These results suggested that HSP47 was closely related to collagens in terms of its expression as well as in its functional relevance.The endoplasmic reticulum (ER) plays important roles in the synthesis and processing of secretory and membranebound proteins. These proteins cotranslationally enter the ER, where they are glycosylated, folded, oligomerized and where some are eventually degraded. Molecular mechanisms describing the regulation of processing and sorting in the ER have, however, not been established (for reviews see Rose and Doms, 1988;Pelham, 1989). Some ER proteins were found to be soluble resident proteins. These proteins include enzymes such as glucosidase I1 (Strous et al, 1987), protein disulfideisomerase (Freedman, 1984) and, more specifically, prolyl-4-hydroxylase for procollagen processing (Kivirikko et al., 1989) that participate in post-translational modification of secretory or membrane-bound proteins, and molecular chaperones such as 78-kDa and 94-kDa glucose-regulated protein (GRP78 and GRP94), thought to retain improperly folded proteins or incompletely assembled protein complexes in the ER (Bole et Correspondence to K. Nagata,

We report the isolation and characterization of a cDNA clone encoding HSP47, a transformation-sensitive heat shock protein that binds to collagen. A cDNA library was prepared from total RNA isolated from heat-shocked chicken embryo fibroblasts and screened by using oligonucleotide mixtures prepared on the basis of the N-terminal amino acid sequence of biochemically purified HSP47. The cDNA insert contained 3,278 bp, which encoded a 15-amino-acid signal peptide and a mature protein coding region consisting of 390 amino acid residues; it also included part of the 5' noncoding region and a long 3' noncoding region. The deduced amino acid sequence revealed an RDEL sequence at the C terminus, which is a variant of the KDEL retention signal for retention of proteins in the endoplasmic reticulum. Northern (RNA) blot analyses and nuclear run-on assays established that the induction of HSP47 by heat shock and its suppression after transformation of chicken embryo fibroblasts by Rous sarcoma virus are regulated at the transcriptional level. A homology search revealed that this protein belongs to the serpin family, the superfamily of plasma serine protease inhibitors. Although structurally homologous to the serpins, HSP47 lacks the active site thought to be essential for the inhibition of proteases and does not appear to bind to intracellular proteases. HSP47 is the first heat shock protein found to be a member of the serpin superfamily. Conversely, it is the first serpin family member that is not secreted from cells, which could be explained by acquisition of the RDEL retention signal during evolution.When living cells of organisms ranging from bacteria to humans are exposed to temperatures 5 to 10°C above the optimum for growth, they respond by synthesizing a group of proteins called heat shock proteins (HSPs). Because these proteins are induced in response to other stresses, such as heavy metals, amino acid analogs, glucose starvation, ethanol, and hypoxia, they are more generally termed stress proteins. In mammalian cells, three major groups of HSPs have been well characterized, the HSP90 and HSP70 families, and a small HSP, ' HSP28 (22,43). In addition to responding to stresses, some of these proteins are synthesized constitutively, and their roles under normal conditions have been shown to be essential for physiological functions, including membrane transport of proteins, as well as folding and unfolding of proteins (9). These functions of stress proteins have led to their designation as molecular chaperones.A novel heat shock glycoprotein of Mr = 47,000 has recently been identified in chicken embryo fibroblasts (CEF), in addition to the well-known HSPs described above (22,43). This heat shock protein (HSP47) is characteristic in its capacity to bind to collagen (28) and its sensitivity to malignant transformation (30, 31). The synthesis of HSP47 is decreased in CEF transformed by Rous sarcoma virus (RSV) and in BALB/c 3T3 cells transformed by simian virus 40 (30, 31). The degree of phosphorylation of HSP47 increase...