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,