The Experts below are selected from a list of 306 Experts worldwide ranked by ideXlab platform
Kazuhiro Nagata - One of the best experts on this subject based on the ideXlab platform.
-
roles of the endoplasmic reticulum resident collagen specific molecular chaperone hsp47 in vertebrate cells and human disease
Journal of Biological Chemistry, 2019Co-Authors: Shinya Ito, Kazuhiro NagataAbstract:Heat shock protein 47 (Hsp47) is an endoplasmic reticulum (ER)-resident molecular chaperone essential for correct folding of Procollagen in mammalian cells. In this Review, we discuss the role and function of Hsp47 in vertebrate cells and its role in connective tissue disorders. Hsp47 binds to collagenous (Gly–Xaa–Arg) repeats within triple-helical Procollagen in the ER and can prevent its local unfolding or aggregate formation, resulting in accelerating triple-helix formation of Procollagen. Hsp47 pH-dependently dissociates from Procollagen in the cis-Golgi or ER–Golgi intermediate compartment and is then transported back to the ER. Although Hsp47 belongs to the serine protease inhibitor (serpin) superfamily, it does not possess serine protease inhibitory activity. Whereas general molecular chaperones such as Hsp70 and Hsp90 exhibit broad substrate specificity, Hsp47 has narrower specificity mainly for Procollagens. However, other Hsp47-interacting proteins have been recently reported, suggesting a much broader role for Hsp47 in the cell that warrants further investigation. Other ER-resident stress proteins, such as binding immunoglobulin protein (BiP), are induced by ER stress, whereas Hsp47 is induced only by heat shock. Constitutive expression of Hsp47 is always correlated with expression of various collagen types, and disruption of the Hsp47 gene in mice causes embryonic lethality due to impaired basement membrane and collagen fibril formation. Increased Hsp47 expression is associated with collagen-related disorders such as fibrosis, characterized by abnormal collagen accumulation, highlighting Hsp47's potential as a clinically relevant therapeutic target.
-
direct in vitro and in vivo evidence for interaction between hsp47 protein and collagen triple helix
Journal of Biological Chemistry, 2012Co-Authors: Takashi Ono, Takahiro Miyazaki, Yoshihito Ishida, Masayoshi Uehata, Kazuhiro NagataAbstract:Hsp47 (heat shock protein 47), a collagen-specific molecular chaperone, is essential for the maturation of various types of Procollagens. Previous studies have suggested that Hsp47 may preferentially recognize the triple-helix form of Procollagen rather than unfolded Procollagen chains in the endoplasmic reticulum. However, the underlying mechanism has remained unclear because of limitations in the available methods for detecting in vitro and in vivo interactions between Hsp47 and collagen. In this study, we established novel methods for this purpose by adopting a time-resolved FRET technique in vitro and a bimolecular fluorescence complementation technique in vivo. Using these methods, we provide direct evidence that Hsp47 binds to collagen triple helices but not to the monomer form in vitro. We also demonstrate that Hsp47 binds a collagen model peptide in the trimer conformation in vivo. Hsp47 did not bind collagen peptides that had been modified to block their ability to form triple helices in vivo. These results conclusively indicate that Hsp47 recognizes the triple-helix form of Procollagen in vitro and in vivo.
-
autophagic elimination of misfolded Procollagen aggregates in the endoplasmic reticulum as a means of cell protection
Molecular Biology of the Cell, 2009Co-Authors: Yoshihito Ishida, Akitsugu Yamamoto, Akira Kitamura, Shireen R Lamande, Tamotsu Yoshimori, John F Bateman, Hiroshi Kubota, Kazuhiro NagataAbstract:Type I collagen is a major component of the extracellular matrix, and mutations in the collagen gene cause several matrix-associated diseases. These mutant Procollagens are misfolded and often aggregated in the endoplasmic reticulum (ER). Although the misfolded Procollagens are potentially toxic to the cell, little is known about how they are eliminated from the ER. Here, we show that Procollagen that can initially trimerize but then aggregates in the ER are eliminated by an autophagy-lysosome pathway, but not by the ER-associated degradation (ERAD) pathway. Inhibition of autophagy by specific inhibitors or RNAi-mediated knockdown of an autophagy-related gene significantly stimulated accumulation of aggregated Procollagen trimers in the ER, and activation of autophagy with rapamycin resulted in reduced amount of aggregates. In contrast, a mutant Procollagen which has a compromised ability to form trimers was degraded by ERAD. Moreover, we found that autophagy plays an essential role in protecting cells against the toxicity of the ERAD-inefficient Procollagen aggregates. The autophagic elimination of aggregated Procollagen occurs independently of the ERAD system. These results indicate that autophagy is a final cell protection strategy deployed against ER-accumulated cytotoxic aggregates that are not able to be removed by ERAD.
-
specific recognition of the collagen triple helix by chaperone hsp47 minimal structural requirement and spatial molecular orientation
Journal of Biological Chemistry, 2006Co-Authors: Takaki Koide, Shinichi Asada, Yoshifumi Takahara, Yoshimi Nishikawa, Kazuhiro Nagata, Kouki KitagawaAbstract:The unique folding of Procollagens in the endoplasmic reticulum is achieved with the assistance of Procollagen-specific molecular chaperones. Heat-shock protein 47 (HSP47) is an endoplasmic reticulum-resident chaperone that plays an essential role in normal Procollagen folding, although its molecular function has not yet been clarified. Recent advances in studies on the binding specificity of HSP47 have revealed that Arg residues at Yaa positions in collagenous Gly-Xaa-Yaa repeats are critical for its interactions (Koide, T., Takahara, Y., Asada, S., and Nagata, K. (2002) J. Biol. Chem. 277, 6178-6182; Tasab, M., Jenkinson, L., and Bulleid, N. J. (2002) J. Biol. Chem. 277, 35007-35012). In the present study, we further examined the client recognition mechanism of HSP47 by taking advantage of systems employing engineered collagen model peptides. First, in vitro binding studies using conformationally constrained collagen-like peptides revealed that HSP47 only recognized correctly folded triple helices and that the interaction with the corresponding single-chain polypeptides was negligible. Second, a binding study using heterotrimeric model clients for HSP47 demonstrated a minimal requirement for the number of Arg residues in the triple helix. Finally, a cross-linking study using photoreactive collagenous peptides provided information about the spatial orientation of an HSP47 molecule in the chaperone-collagen complex. The obtained results led to the development of a new model of HSP47-collagen complexes that differs completely from the previously proposed "flying capstan model" (Dafforn, T. R., Della, M., and Miller, A. D. (2001) J. Biol. Chem. 276, 49310-49319).
-
intracellular interaction of collagen specific stress protein hsp47 with newly synthesized Procollagen
Journal of Cell Biology, 1996Co-Authors: Mamoru Satoh, Kazunori Hirayoshi, S I Yokota, Nobuko Hosokawa, Kazuhiro NagataAbstract:Heat shock protein 47 (HSP47), a collagen-specific 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 alpha, alpha'-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 endoglycosidase 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.
Darwin J. Prockop - One of the best experts on this subject based on the ideXlab platform.
-
recombinant Procollagen ii deletion of d period segments identifies sequences that are required for helix stabilization and generates a temperature sensitive n proteinase cleavage site
Journal of Biological Chemistry, 1998Co-Authors: William V Arnold, Hisashi Hattori, Diane Mechling, Andrzej Fertala, Aleksander Sieron, Hans Peter Bachinger, Darwin J. ProckopAbstract:Abstract A cDNA cassette system was used to synthesize recombinant versions of Procollagen II in which one of the four blocks of 234 amino acids that define a repeating D periods of the collagen triple helix were deleted. All the proteins were triple helical and all underwent a helix-to-coil transition between 25 and 42 °C as assayed by circular dichroism. However, the details of the melting curves varied. The Procollagen lacking the D1 period unfolded 3 °C lower than a full-length molecule. With the Procollagen lacking the D4 period, the first 25% of unfolding occurred at a lower temperature than the full-length molecule, but the rest of the structure unfolded at the same temperature. With the Procollagen lacking the terminal D0.4 period, the protein unfolded 3 °C lower than the full-length molecule and a smaller fraction of the protein was secreted by stably transfected clones than with the other recombinant Procollagens. The results confirmed previous suggestions that the collagen triple helix contains regions of varying stability and they demonstrated that the two D periods at the end of the molecule contain sequences that serve as clamps for folding and for stabilizing the triple helix. Reaction of the recombinant Procollagens with Procollagen N-proteinase indicated that in the Procollagen lacking the sequences, the D1 period assumed an unusual temperature-sensitive conformation at 35 °C that allowed cleavage at an otherwise resistant Gly-Ala bond between residues 394 and 395 of the α1(II) chain.
-
Procollagen n proteinase and Procollagen c proteinase two unusual metalloproteinases that are essential for Procollagen processing probably have important roles in development and cell signaling
Matrix Biology, 1998Co-Authors: Darwin J. Prockop, Aleksander Sieron, Shiwu LiAbstract:Abstract As soon as Procollagen precursors of fibrillar collagens were discovered in the early 1970s, it became apparent that connective tissues must contain proteolytic activities that cleave the N-propeptides and the C-propeptides from Procollagens. Isolation and characterization of the enzymic activities, however, proved to be unexpected difficult. Both proteinases are large and are synthesized in several different forms with polypeptide chains ranging in size from 70 kDa to about 130 kDa. The N-proteinase has the unusual property of cleaving the N-propeptides from type I and type II Procollagens if the proteins are in a native conformation, but not if the proteins are partially unfolded so that the N-telopeptides are no longer in a hair-pin configuration. The C-proteinase specifically cleaves native and denatured types I, II and III Procollagens. It also specifically cleaves a precursor of lysyl oxidase and laminin 5. Both enzymes and their variants have structures that place them in a large and expanding super-family of over 200 zinc-binding metalloproteinases. The larger of two forms of the N-proteinase contains an RGD sequence for binding through integrins and properdin repeats similar to those found in thrombospondin. The shorter 70 kDa form of the C-proteinase is identical to the protein that was previously identified as bone morphogenic protein-1. Both the 70 kDa C-proteinase and two larger forms are homologous to proteins that are expressed early in development in a variety of organisms, including Drosophila, sea urchin, and fish. Therefore, the data suggest that both the N- and C-proteinases have important biological functions in addition to the roles in the processing of Procollagens.
-
cDNA cloning and expression of bovine Procollagen I N-proteinase: A new member of the superfamily of zinc-metalloproteinases with binding sites for cells and other matrix components
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Alain Colige, Shiwu Li, Betty Nusgens, Darwin J. Prockop, Aleksander Sieron, Charles M LapiereAbstract:Procollagen N-proteinase (EC 3.4.24.14) cleaves the amino-propeptides in the processing of type I and type II Procollagens to collagens. Deficiencies of the enzyme cause dermatosparaxis in cattle and sheep, and they cause type VIIC Ehlers–Danlos syndrome in humans, heritable disorders characterized by accumulation of pNcollagen and severe skin fragility. Amino acid sequences for the N-proteinase were used to obtain cDNAs from bovine skin. Three overlapping cDNAs had an ORF coding for a protein of 1205 residues. Mammalian cells stably transfected with a complete cDNA secreted an active recombinant enzyme that specifically cleaved type I Procollagen. The protein contained zinc-binding sequences of the clan MB of metallopeptidases that includes Procollagen C-proteinase/BMP-1. The protein also contained four repeats that are homologous to domains found in thrombospondins and in properdin and that can participate in complex intermolecular interactions such as activation of latent forms of transforming growth factor β or the binding to sulfatides. Therefore, the enzyme may play a role in development that is independent of its role in collagen biosynthesis. This hypothesis was supported by the observation that in some tissues the levels of mRNA for the enzyme are disproportionately high relative to the apparent rate of collagen biosynthesis.
-
the c proteinase that processes Procollagens to fibrillar collagens is identical to the protein previously identified as bone morphogenic protein 1
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Shiwu Li, Yoshio Hojima, William V Arnold, Andrzej Fertala, Aleksander Sieron, Darwin J. ProckopAbstract:Abstract Bone morphogenic protein-1 (BMP-1) was originally identified as one of several BMPs that induced new bone formation when implanted into ectopic sites in rodents. BMP-1, however, differed from other BMPs in that it its structure was not similar to transforming growth factor beta. Instead, it had a large domain homologous to a metalloendopeptidase isolated from crayfish, an epidermal growth-factor-like domain, and three regions of internal sequence homology referred to as CUB domains. Therefore, BMP-1 was a member of the "astacin families" of zinc-requiring endopeptidases. Many astacins have been shown to play critical roles in embryonic hatching, dorsal/ventral patterning, and early developmental decisions. Here, we have obtained amino acid sequences and isolated cDNA clones for Procollagen C-proteinase (EC 3.4.24.19), an enzyme that is essential for the processing of Procollagens to fibrillar collagens. The results demonstrate that Procollagen C-proteinase is identical to BMP-1.
-
characterization of type i Procollagen n proteinase from fetal bovine tendon and skin purification of the 500 kilodalton form of the enzyme from bovine tendon
Journal of Biological Chemistry, 1994Co-Authors: Yoshio Hojima, Anne M Romanic, J A Mckenzie, M M Morgelin, Margueritemarie Boutillon, Gichung Chen, N Rafi, Jürgen Engel, Michel Van Der Rest, Darwin J. ProckopAbstract:Abstract Procollagen N-proteinase (EC 3.4.24.14) is the enzyme that specifically cleaves the NH2-terminal propeptides from type I Procollagen. Two forms of N-proteinase with apparent molecular sizes of 300 and 500 kDa were found in partially purified preparations from fetal bovine tendon extracts. The 500-kDa form of enzyme was purified 16,000-fold with a recovery of 8% from the extracts of the tendons by six purification steps. The purified enzyme was a neutral, Ca(2+)-dependent proteinase (5-10 mM) that was inhibited by metal chelators. The 500-kDa enzyme contained unreduced polypeptides of 58, 125, 170, and 190 kDa which were separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Electron microscopic study indicated that the enzyme molecules were generally globular and had diameters of 33 +/- 4 nm. Other properties of the 500-kDa enzyme were: 1) the Km for type I Procollagen is 35 nM at pH 7.5 and 35 degrees C, and the kcat is 290 h-1; 2) the activation energy for reaction with type I Procollagen is 10,050 cal mol-1; 3) the isoelectric point is 3.8; 4) the enzyme cleaves the NH2-terminal propeptides of type II Procollagen as well as type I Procollagen but not of type III Procollagen; and 5) the enzyme specifically cleaves a -Pro-Gln- bond in the pro-alpha 1(I) chain and an -Ala-Gln- bond in the pro-alpha 2(I) chain. The bovine N-proteinase with a mass of 300 kDa was found to be similar to the 500-kDa enzyme and appeared to be a degraded form of the 500-kDa enzyme generated during purification. The N-proteinase from fetal bovine skin extracts also contained 300-kDa and 500-kDa enzyme forms.
Gary J Fisher - One of the best experts on this subject based on the ideXlab platform.
-
reduced expression of connective tissue growth factor ctgf ccn2 mediates collagen loss in chronologically aged human skin
Journal of Investigative Dermatology, 2010Co-Authors: Taihao Quan, Tianyuan He, Yuan Shao, John J Voorhees, Gary J FisherAbstract:Reduced production of type I Procollagen is a prominent feature of chronologically aged human skin. Connective tissue growth factor (CTGF/CCN2), a downstream target of the transforming growth factor-β (TGF-β)/Smad pathway, is highly expressed in numerous fibrotic disorders, in which it is believed to stimulate excessive collagen production. CTGF is constitutively expressed in normal human dermis in vivo, suggesting that CTGF is a physiological regulator of collagen expression. We report here that the TGF-β/Smad/CTGF axis is significantly reduced in dermal fibroblasts, the major collagen-producing cells, in aged (≥80 years) human skin in vivo. In primary human skin fibroblasts, neutralization of endogenous TGF-β or knockdown of CTGF substantially reduced the expression of type I Procollagen mRNA, protein, and promoter activity. In contrast, overexpression of CTGF stimulated type I Procollagen expression, and increased promoter activity. Inhibition of TGF-β receptor kinase, knockdown of Smad4, or overexpression of inhibitory Smad7 abolished CTGF stimulation of type I Procollagen expression. However, CTGF did not stimulate Smad3 phosphorylation or Smad3-dependent transcriptional activity. These data indicate that in human skin fibroblasts, type I Procollagen expression is dependent on endogenous production of both TGF-β and CTGF, which act through interdependent yet distinct mechanisms. Downregulation of the TGF-β/Smad/CTGF axis likely mediates reduced type I Procollagen expression in aged human skin in vivo.
-
connective tissue remodeling induced by carbon dioxide laser resurfacing of photodamaged human skin
Archives of Dermatology, 2004Co-Authors: Jeffrey S Orringer, Ted A. Hamilton, Timothy M Johnson, Darius J Karimipour, Craig Hammerberg, Sewon Kang, John J Voorhees, Gary J FisherAbstract:Objective To quantitatively examine the dynamics of molecular alterations involved in dermal remodeling after carbon dioxide (CO 2 ) laser resurfacing of photodamaged human skin. Design Serial in vivo biochemical analyses after laser therapy. Setting Academic referral center, Department of Dermatology, University of Michigan, Ann Arbor. Subjects Volunteer sample of 28 adults, 48 to 76 years old, with clinically evident photodamage of the forearms. Intervention Focal CO 2 laser resurfacing of photodamaged forearms and serial biopsies at baseline and various times after treatment. Main Outcome Measures Reverse transcriptase real-time polymerase chain reaction technology and immunohistochemistry were used to assess levels of type I and type III Procollagens; matrix metalloproteinases (MMPs) 1, 3, 9, and 13; tropoelastin; fibrillin; primary cytokines interleukin 1β and tumor necrosis factor α; and profibrotic cytokine transforming growth factor β1. Results Production of type I Procollagen and type III Procollagen messenger RNA peaked at 7.5 and 8.9 times baseline levels, respectively, 21 days after treatment and remained elevated for at least 6 months. Increases in messenger RNA levels of several cytokines (interleukin 1β, tumor necrosis factor α, and transforming growth factor β1) preceded and/or accompanied changes in collagen levels. Marked increases in messenger RNA levels of MMP-1 (39 130-fold), MMP-3 (1041-fold), MMP-9 (75-fold), and MMP-13 (767-fold) were noted. Levels of fibrillin and tropoelastin rose in a delayed fashion several weeks after treatment. Conclusions The biochemical changes seen after CO 2 laser resurfacing proceed through a well-organized and highly reproducible wound healing response that results in marked alterations in dermal structure. These quantitative changes may serve as a means for comparison as other therapeutic modalities meant to improve the appearance of photodamaged skin are evaluated.
-
Solar ultraviolet irradiation reduces collagen in photoaged human skin by blocking transforming growth factor-β type II receptor/Smad signaling
American Journal of Pathology, 2004Co-Authors: Taihao Quan, Tianyuan He, Sewon Kang, John J Voorhees, Gary J FisherAbstract:Ultraviolet (UV) irradiation from the sun reduces production of type I Procollagen (COLI), the major structural protein in toman skin. This reduction is a key feature of the pathophysiology of premature skin aging (photoaging). Photoaging is the most common form of skin damage and is associated with skin carcinoma. TGF-β/Smad pathway is the major regulator of type I Procollagen synthesis in human skin. We have previously reported that UV irradiation impairs transforming growth factor-β (TGF-β)/Smad signaling in mink lung epithelial cells. We have investigated the mechanism of UV irradiation impairment of the TGf-β/Smad pathway and die impact of this impairment on type I Procollagen production in human skin fibroblasts, the major collagen-producing cells in skin. We report here that UV irradiation impairs TGF-β/Smad pathway in human skin by down-regulation of TGF-β type receptor (TβRII). This loss of TβRII occurs within 8 hours after UV irradiation and precedes down-regulation of type I Procollagen expression in human skin, in vivo. In human skin fibroblasts, UV-induced TβRII down-regulation is mediated by transcriptional repression and results in 90% reduction of specific, cell-surface binding of TGF-β. This loss of TβRII prevents downstream activation of Smad2/3 by TGF-β, thereby reducing expression of type I Procollagen. Preventing loss of TβRII by overexpression protects against UV inhibition of type I Procollagen gene expression hi human skin fibroblasts. UV-induced down-regulation of TβRII, with attendant reduction of type I Procollagen production, is a critical molecular mechanism in the pathophysiology of photoaging.
-
inhibition of type i Procollagen production in photodamage correlation between presence of high molecular weight collagen fragments and reduced Procollagen synthesis
Journal of Investigative Dermatology, 2002Co-Authors: James Varani, Patricia Perone, Suzanne E. G. Fligiel, Gary J Fisher, John J VoorheesAbstract:Three-dimensional lattices of reconstituted, polymerized type I collagen were subjected to partial hydrolysis by organ culture fluid from human skin or by various matrix metalloproteinases, including matrix metalloproteinase-1 (interstitial collagenase), -2 (72 kDa gelatinase A), -8 (neutrophil collagenase), -9 (92 kDa gelatinase B), or -13 (collagenase 3). Following partial digestion, human dermal fibroblasts were incubated on the enzyme-treated or control lattices and examined for ability to contract the collagen lattice and synthesize type I Procollagen. Collagen lattices partially degraded by organ culture fluid were contracted by fibroblasts under conditions in which control collagen lattices were not. On the partially degraded collagen, fibroblasts synthesized reduced amounts of type I Procollagen (approximately 70% reduction). Purified matrix metalloproteinases with collagenolytic activity duplicated the effects of the human skin organ culture fluid, although matrix metalloproteinases 8 and 13 were less efficient than matrix metalloproteinase-1 (65% vs 40% and 18% reduction in type I Procollagen production for matrix metalloproteinases 1, 8, and 13, respectively). Matrix metalloproteinases 2 and 9 were without effect on intact collagen; however, when collagen lattices were subjected to digestion by a combination of matrix metalloproteinases 1 and 9, fragments produced by matrix metalloproteinase-1 were further degraded by the gelatinase. Collagen contraction and inhibition of Procollagen synthesis were both reduced. Matrix metalloproteinase-2 was less effective than matrix metalloproteinase-9 in clearing matrix metalloproteinase-1-generated fragments. Matrix metalloproteinase-2 was also less effective in preventing contraction and inhibiting the downregulation of type I Procollagen synthesis. These observations suggest that in the presence of high molecular weight fragments of type I collagen, type I Procollagen synthesis is inhibited. As these fragments are processed further, there is less inhibition of type I Procollagen production.
-
inhibition of type i Procollagen synthesis by damaged collagen in photoaged skin and by collagenase degraded collagen in vitro
American Journal of Pathology, 2001Co-Authors: James Varani, Zeng Quan Wang, Yuan Shao, Dara Spearman, Patricia Perone, Suzanne E. G. Fligiel, Gary J Fisher, Subhash C Datta, Sewon Kang, John J VoorheesAbstract:Type I and type III Procollagen are reduced in photodamaged human skin. This reduction could result from increased degradation by metalloproteinases and/or from reduced Procollagen synthesis. In the present study, we investigated type I Procollagen production in photodamaged and sun-protected human skin. Skin samples from severely sun-damaged forearm skin and matched sun-protected hip skin from the same individuals were assessed for type I Procollagen gene expression by in situ hybridization and for type I Procollagen protein by immunostaining. Both mRNA and protein were reduced (∼65 and 57%, respectively) in photodamaged forearm skin compared to sun-protected hip skin. We next investigated whether reduced type I Procollagen production was because of inherently reduced capacity of skin fibroblasts in severely photodamaged forearm skin to synthesize Procollagen, or whether contextual influences within photodamaged skin act to down-regulate type I Procollagen synthesis. For these studies, fibroblasts from photodamaged skin and matched sun-protected skin were established in culture. Equivalent numbers of fibroblasts were isolated from the two skin sites. Fibroblasts from the two sites had similar growth capacities and produced virtually identical amounts of type I Procollagen protein. These findings indicate that the lack of type I Procollagen synthesis in sun-damaged skin is not because of irreversible damage to fibroblast collagen-synthetic capacity. It follows, therefore, that factors within the severely photodamaged skin may act in some manner to inhibit Procollagen production by cells that are inherently capable of doing so. Interactions between fibroblasts and the collagenous extracellular matrix regulate type I Procollagen synthesis. In sun-protected skin, collagen fibrils exist as a highly organized matrix. Fibroblasts are found within the matrix, in close apposition with collagen fibers. In photodamaged skin, collagen fibrils are shortened, thinned, and disorganized. The level of partially degraded collagen is ∼3.6-fold greater in photodamaged skin than in sun-protected skin, and some fibroblasts are surrounded by debris. To model this situation, skin fibroblasts were cultured in vitro on intact collagen or on collagen that had been partially degraded by exposure to collagenolytic enzymes. Collagen that had been partially degraded by exposure to collagenolytic enzymes from either bacteria or human skin underwent contraction in the presence of dermal fibroblasts, whereas intact collagen did not. Fibroblasts cultured on collagen that had been exposed to either source of collagenolytic enzyme demonstrated reduced proliferative capacity (22 and 17% reduction on collagen degraded by bacterial collagenase or human skin collagenase, respectively) and synthesized less type I Procollagen (36 and 88% reduction, respectively, on a per cell basis). Taken together, these findings indicate that 1) fibroblasts from photoaged and sun-protected skin are similar in their capacities for growth and type I Procollagen production; and 2) the accumulation of partially degraded collagen observed in photodamaged skin may inhibit, by an as yet unidentified mechanism, type I Procollagen synthesis.
Linda J. Sandell - One of the best experts on this subject based on the ideXlab platform.
-
reexpression of type iia Procollagen by adult articular chondrocytes in osteoarthritic cartilage
Arthritis & Rheumatism, 1999Co-Authors: Thomas Aigner, Howard H Chansky, Frederick A Matsen, William J Maloney, Linda J. SandellAbstract:Objective. To test for the reexpression of the chondroprogenitor splice variant of the gene COL2A1, type IIA Procollagen (containing a cysteine-rich NH 2 propeptide), in adult articular chondrocytes in osteoarthritic (OA) joint disease. Methods. In situ hybridization and immunohistochemical localization were performed on normal and OA articular cartilage specimens. The presence of type IIA Procollagen messenger RNA (mRNA) expression was confirmed by Northern blot analysis. Results. In normal articular cartilage, no expression of mRNA or presence of type IIA Procollagen was found. In OA articular cartilage, focally intense staining for type IIA protein was detected. Consistent with this, chondrocytes, particularly in the middle zones of articular cartilage, expressed type IIA Procollagen mRNA. OA repair cartilage typically showed a broad zone of cells expressing type IIA mRNA and protein. Conclusion. Type IIA Procollagen is reexpressed by adult articular chondrocytes in OA cartilage degeneration, indicating the potential reversion of the cells to a chondroprogenitor cellular phenotype. The absence of type IIA mRNA and protein in normal adult articular cartilage and its onset in the diseased state suggests type IIA Procollagen as a marker of OA.
-
type iia Procollagen containing the cysteine rich amino propeptide is deposited in the extracellular matrix of prechondrogenic tissue and binds to tgf β1 and bmp 2
Journal of Cell Biology, 1999Co-Authors: Yong Zhu, Anush Oganesian, Douglas R Keene, Linda J. SandellAbstract:Type II Procollagen is expressed as two splice forms. One form, type IIB, is synthesized by chondrocytes and is the major extracellular matrix component of cartilage. The other form, type IIA, contains an additional 69 amino acid cysteine-rich domain in the NH2-propeptide and is synthesized by chondrogenic mesenchyme and perichondrium. We have hypothesized that the additional protein domain of type IIA Procollagen plays a role in chondrogenesis. The present study was designed to determine the localization of the type IIA NH2-propeptide and its function during chondrogenesis. Immunofluorescence histochemistry using antibodies to three domains of the type IIA Procollagen molecule was used to localize the NH2-propeptide, fibrillar domain, and COOH-propeptides of the type IIA Procollagen molecule during chondrogenesis in a developing human long bone (stage XXI). Before chondrogenesis, type IIA Procollagen was synthesized by chondroprogenitor cells and deposited in the extracellular matrix. Immunoelectron microscopy revealed type IIA Procollagen fibrils labeled with antibodies to NH2-propeptide at ∼70 nm interval suggesting that the NH2-propeptide remains attached to the collagen molecule in the extracellular matrix. As differentiation proceeds, the cells switch synthesis from type IIA to IIB Procollagen, and the newly synthesized type IIB collagen displaces the type IIA Procollagen into the interterritorial matrix. To initiate studies on the function of type IIA Procollagen, binding was tested between recombinant NH2-propeptide and various growth factors known to be involved in chondrogenesis. A solid phase binding assay showed no reaction with bFGF or IGF-1, however, binding was observed with TGF-β1 and BMP-2, both known to induce endochondral bone formation. BMP-2, but not IGF-1, coimmunoprecipitated with type IIA NH2-propeptide. Recombinant type IIA NH2-propeptide and type IIA Procollagen from media coimmunoprecipitated with BMP-2 while recombinant type IIB NH2-propeptide and all other forms of type II Procollagens and mature collagen did not react with BMP-2. Taken together, these results suggest that the NH2-propeptide of type IIA Procollagen could function in the extracellular matrix distribution of bone morphogenetic proteins in chondrogenic tissue.
-
alternatively spliced type ii Procollagen mrnas define distinct populations of cells during vertebral development differential expression of the amino propeptide
Journal of Cell Biology, 1991Co-Authors: Linda J. Sandell, N Morris, J R Robbins, Mary B GoldringAbstract:Type II collagen is a major component of cartilage providing structural integrity to the tissue. Type II Procollagen can be expressed in two forms by differential splicing of the primary gene transcript. The two mRNAs either include (type IIA) or exclude (type IIB) an exon (exon 2) encoding the major portion of the amino (NH2)-propeptide (Ryan, M. C., and L. J. Sandell. 1990. J. Biol. Chem. 265:10334-10339). The expression of the two Procollagens was examined in order to establish a potential functional significance for the two type II Procollagen mRNAs. First, to establish whether the two mRNAs are functional, we showed that both mRNAs can be translated and the proteins secreted into the extracellular environment. Both proteins were identified as type II Procollagens. Secondly, to test the hypothesis that differential expression of type II Procollagens may be a marker for a distinct population of cells, specific Procollagen mRNAs were localized in tissue by in situ hybridization to oligonucleotides spanning the exon junctions. Embryonic vertebral column was chosen as a source of tissue undergoing rapid chondrogenesis, allowing the examination of a variety of cell types related to cartilage. In this issue, each Procollagen mRNA had a distinct tissue distribution during chondrogenesis with type IIB expressed in chondrocytes and type IIA expressed in cells surrounding cartilage in prechondrocytes. The morphology of the cells expressing the two collagen types was distinct: the cells expressing type IIA are narrow, elongated, and "fibroblastic" in appearance while the cells expressing type IIB are large and round. The expression of type IIB appears to be correlated with abundant synthesis and accumulation of cartilagenous extracellular matrix. The expression of type IIB is spatially correlated with the high level expression of the cartilage proteoglycan, aggrecan, establishing type IIB Procollagen and aggrecan as markers for the chondrocyte phenotype. Transcripts of type II collagen, primarily type IIA, are also expressed in embryonic spinal ganglion. While small amounts of type II collagen have been previously detected in noncartilagenous tissues, the detection of this new form of the collagen in relatively high abundance in embryonic nerve tissue is unique. Taken together, these findings imply a potential functional difference between type IIA and type IIB Procollagens and indicate that the removal of exon 2 from the pre-mRNA, and consequently the NH2-propeptide from the collagen molecule, may be an important step in chondrogenesis. In addition, type II Procollagen, specifically type IIA, may function in noncartilage tissues, particularly during development.
John J Voorhees - One of the best experts on this subject based on the ideXlab platform.
-
reduced expression of connective tissue growth factor ctgf ccn2 mediates collagen loss in chronologically aged human skin
Journal of Investigative Dermatology, 2010Co-Authors: Taihao Quan, Tianyuan He, Yuan Shao, John J Voorhees, Gary J FisherAbstract:Reduced production of type I Procollagen is a prominent feature of chronologically aged human skin. Connective tissue growth factor (CTGF/CCN2), a downstream target of the transforming growth factor-β (TGF-β)/Smad pathway, is highly expressed in numerous fibrotic disorders, in which it is believed to stimulate excessive collagen production. CTGF is constitutively expressed in normal human dermis in vivo, suggesting that CTGF is a physiological regulator of collagen expression. We report here that the TGF-β/Smad/CTGF axis is significantly reduced in dermal fibroblasts, the major collagen-producing cells, in aged (≥80 years) human skin in vivo. In primary human skin fibroblasts, neutralization of endogenous TGF-β or knockdown of CTGF substantially reduced the expression of type I Procollagen mRNA, protein, and promoter activity. In contrast, overexpression of CTGF stimulated type I Procollagen expression, and increased promoter activity. Inhibition of TGF-β receptor kinase, knockdown of Smad4, or overexpression of inhibitory Smad7 abolished CTGF stimulation of type I Procollagen expression. However, CTGF did not stimulate Smad3 phosphorylation or Smad3-dependent transcriptional activity. These data indicate that in human skin fibroblasts, type I Procollagen expression is dependent on endogenous production of both TGF-β and CTGF, which act through interdependent yet distinct mechanisms. Downregulation of the TGF-β/Smad/CTGF axis likely mediates reduced type I Procollagen expression in aged human skin in vivo.
-
connective tissue remodeling induced by carbon dioxide laser resurfacing of photodamaged human skin
Archives of Dermatology, 2004Co-Authors: Jeffrey S Orringer, Ted A. Hamilton, Timothy M Johnson, Darius J Karimipour, Craig Hammerberg, Sewon Kang, John J Voorhees, Gary J FisherAbstract:Objective To quantitatively examine the dynamics of molecular alterations involved in dermal remodeling after carbon dioxide (CO 2 ) laser resurfacing of photodamaged human skin. Design Serial in vivo biochemical analyses after laser therapy. Setting Academic referral center, Department of Dermatology, University of Michigan, Ann Arbor. Subjects Volunteer sample of 28 adults, 48 to 76 years old, with clinically evident photodamage of the forearms. Intervention Focal CO 2 laser resurfacing of photodamaged forearms and serial biopsies at baseline and various times after treatment. Main Outcome Measures Reverse transcriptase real-time polymerase chain reaction technology and immunohistochemistry were used to assess levels of type I and type III Procollagens; matrix metalloproteinases (MMPs) 1, 3, 9, and 13; tropoelastin; fibrillin; primary cytokines interleukin 1β and tumor necrosis factor α; and profibrotic cytokine transforming growth factor β1. Results Production of type I Procollagen and type III Procollagen messenger RNA peaked at 7.5 and 8.9 times baseline levels, respectively, 21 days after treatment and remained elevated for at least 6 months. Increases in messenger RNA levels of several cytokines (interleukin 1β, tumor necrosis factor α, and transforming growth factor β1) preceded and/or accompanied changes in collagen levels. Marked increases in messenger RNA levels of MMP-1 (39 130-fold), MMP-3 (1041-fold), MMP-9 (75-fold), and MMP-13 (767-fold) were noted. Levels of fibrillin and tropoelastin rose in a delayed fashion several weeks after treatment. Conclusions The biochemical changes seen after CO 2 laser resurfacing proceed through a well-organized and highly reproducible wound healing response that results in marked alterations in dermal structure. These quantitative changes may serve as a means for comparison as other therapeutic modalities meant to improve the appearance of photodamaged skin are evaluated.
-
Solar ultraviolet irradiation reduces collagen in photoaged human skin by blocking transforming growth factor-β type II receptor/Smad signaling
American Journal of Pathology, 2004Co-Authors: Taihao Quan, Tianyuan He, Sewon Kang, John J Voorhees, Gary J FisherAbstract:Ultraviolet (UV) irradiation from the sun reduces production of type I Procollagen (COLI), the major structural protein in toman skin. This reduction is a key feature of the pathophysiology of premature skin aging (photoaging). Photoaging is the most common form of skin damage and is associated with skin carcinoma. TGF-β/Smad pathway is the major regulator of type I Procollagen synthesis in human skin. We have previously reported that UV irradiation impairs transforming growth factor-β (TGF-β)/Smad signaling in mink lung epithelial cells. We have investigated the mechanism of UV irradiation impairment of the TGf-β/Smad pathway and die impact of this impairment on type I Procollagen production in human skin fibroblasts, the major collagen-producing cells in skin. We report here that UV irradiation impairs TGF-β/Smad pathway in human skin by down-regulation of TGF-β type receptor (TβRII). This loss of TβRII occurs within 8 hours after UV irradiation and precedes down-regulation of type I Procollagen expression in human skin, in vivo. In human skin fibroblasts, UV-induced TβRII down-regulation is mediated by transcriptional repression and results in 90% reduction of specific, cell-surface binding of TGF-β. This loss of TβRII prevents downstream activation of Smad2/3 by TGF-β, thereby reducing expression of type I Procollagen. Preventing loss of TβRII by overexpression protects against UV inhibition of type I Procollagen gene expression hi human skin fibroblasts. UV-induced down-regulation of TβRII, with attendant reduction of type I Procollagen production, is a critical molecular mechanism in the pathophysiology of photoaging.
-
inhibition of type i Procollagen production in photodamage correlation between presence of high molecular weight collagen fragments and reduced Procollagen synthesis
Journal of Investigative Dermatology, 2002Co-Authors: James Varani, Patricia Perone, Suzanne E. G. Fligiel, Gary J Fisher, John J VoorheesAbstract:Three-dimensional lattices of reconstituted, polymerized type I collagen were subjected to partial hydrolysis by organ culture fluid from human skin or by various matrix metalloproteinases, including matrix metalloproteinase-1 (interstitial collagenase), -2 (72 kDa gelatinase A), -8 (neutrophil collagenase), -9 (92 kDa gelatinase B), or -13 (collagenase 3). Following partial digestion, human dermal fibroblasts were incubated on the enzyme-treated or control lattices and examined for ability to contract the collagen lattice and synthesize type I Procollagen. Collagen lattices partially degraded by organ culture fluid were contracted by fibroblasts under conditions in which control collagen lattices were not. On the partially degraded collagen, fibroblasts synthesized reduced amounts of type I Procollagen (approximately 70% reduction). Purified matrix metalloproteinases with collagenolytic activity duplicated the effects of the human skin organ culture fluid, although matrix metalloproteinases 8 and 13 were less efficient than matrix metalloproteinase-1 (65% vs 40% and 18% reduction in type I Procollagen production for matrix metalloproteinases 1, 8, and 13, respectively). Matrix metalloproteinases 2 and 9 were without effect on intact collagen; however, when collagen lattices were subjected to digestion by a combination of matrix metalloproteinases 1 and 9, fragments produced by matrix metalloproteinase-1 were further degraded by the gelatinase. Collagen contraction and inhibition of Procollagen synthesis were both reduced. Matrix metalloproteinase-2 was less effective than matrix metalloproteinase-9 in clearing matrix metalloproteinase-1-generated fragments. Matrix metalloproteinase-2 was also less effective in preventing contraction and inhibiting the downregulation of type I Procollagen synthesis. These observations suggest that in the presence of high molecular weight fragments of type I collagen, type I Procollagen synthesis is inhibited. As these fragments are processed further, there is less inhibition of type I Procollagen production.
-
inhibition of type i Procollagen synthesis by damaged collagen in photoaged skin and by collagenase degraded collagen in vitro
American Journal of Pathology, 2001Co-Authors: James Varani, Zeng Quan Wang, Yuan Shao, Dara Spearman, Patricia Perone, Suzanne E. G. Fligiel, Gary J Fisher, Subhash C Datta, Sewon Kang, John J VoorheesAbstract:Type I and type III Procollagen are reduced in photodamaged human skin. This reduction could result from increased degradation by metalloproteinases and/or from reduced Procollagen synthesis. In the present study, we investigated type I Procollagen production in photodamaged and sun-protected human skin. Skin samples from severely sun-damaged forearm skin and matched sun-protected hip skin from the same individuals were assessed for type I Procollagen gene expression by in situ hybridization and for type I Procollagen protein by immunostaining. Both mRNA and protein were reduced (∼65 and 57%, respectively) in photodamaged forearm skin compared to sun-protected hip skin. We next investigated whether reduced type I Procollagen production was because of inherently reduced capacity of skin fibroblasts in severely photodamaged forearm skin to synthesize Procollagen, or whether contextual influences within photodamaged skin act to down-regulate type I Procollagen synthesis. For these studies, fibroblasts from photodamaged skin and matched sun-protected skin were established in culture. Equivalent numbers of fibroblasts were isolated from the two skin sites. Fibroblasts from the two sites had similar growth capacities and produced virtually identical amounts of type I Procollagen protein. These findings indicate that the lack of type I Procollagen synthesis in sun-damaged skin is not because of irreversible damage to fibroblast collagen-synthetic capacity. It follows, therefore, that factors within the severely photodamaged skin may act in some manner to inhibit Procollagen production by cells that are inherently capable of doing so. Interactions between fibroblasts and the collagenous extracellular matrix regulate type I Procollagen synthesis. In sun-protected skin, collagen fibrils exist as a highly organized matrix. Fibroblasts are found within the matrix, in close apposition with collagen fibers. In photodamaged skin, collagen fibrils are shortened, thinned, and disorganized. The level of partially degraded collagen is ∼3.6-fold greater in photodamaged skin than in sun-protected skin, and some fibroblasts are surrounded by debris. To model this situation, skin fibroblasts were cultured in vitro on intact collagen or on collagen that had been partially degraded by exposure to collagenolytic enzymes. Collagen that had been partially degraded by exposure to collagenolytic enzymes from either bacteria or human skin underwent contraction in the presence of dermal fibroblasts, whereas intact collagen did not. Fibroblasts cultured on collagen that had been exposed to either source of collagenolytic enzyme demonstrated reduced proliferative capacity (22 and 17% reduction on collagen degraded by bacterial collagenase or human skin collagenase, respectively) and synthesized less type I Procollagen (36 and 88% reduction, respectively, on a per cell basis). Taken together, these findings indicate that 1) fibroblasts from photoaged and sun-protected skin are similar in their capacities for growth and type I Procollagen production; and 2) the accumulation of partially degraded collagen observed in photodamaged skin may inhibit, by an as yet unidentified mechanism, type I Procollagen synthesis.
