Elastic fiber assembly is disrupted by excessive accumulation of chondroitin sulfate in the human dermal fibrotic disease, keloid

Keloid is a fibrotic disease characterized by abnormal accumulation of extracellular matrix in the dermis. The keloid matrix contains excess collagen and glycosaminoglycans (GAGs), but lacks elastic fiber. However, the roles of these matrix components in the pathogenesis of keloid are largely unknown. Here, we show that elastin and DANCE (also known as fibulin-5), a protein required for elastic fiber formation, are not deposited in the extracellular matrix of keloids, due to excess accumulation of chondoitin sulfate (CS), although the expression of elastin and DANCE is not affected. Amount of CS accumulated in the keloid legion was 6.9-fold higher than in normal skin. Fibrillin-1, a scaffold protein for elastic fiber assembly, was abnormally distributed in the keloid matrix. Addition of purified CS to keloid fibroblast culture resulted in abnormal deposition of fibrillin-1, concomitant with significantly decreased accumulation of elastin and DANCE in the extracellular matrix. We propose that CS plays a crucial role in the development of keloid lesions through inhibition of elastic fiber assembly.     

Presence of type III collagen in guinea-pig dermal scar.

Guinea-pig dermal scar was shown to contain type III collagen, and, from densitometric analysis of gel electrophoretograms, it was shown to have a higher concentration than the surrounding dermis. This finding is consistent with the 'embryonic' nature of newly formed dermal wound tissue, reflected in increased hydroxylation of collagen lysine and the presence of dihydroxylysinonorleucine (after reduction) as the major cross-link.     

Cloning,characterization, and functional studies of a 47-kDa platelet receptor for type III collagen

A 1.2-kb cDNA fragment encoding a platelet 47-kDa protein has been isolated from a human bone marrow cDNA library by using a degenerate oligonucleotide of the sequenced amino terminus of the purified platelet protein with a poly(dT)(12).(dG) by polymerase chain reaction. A computer search revealed that the cDNA represents the coding sequence of a protein with a fragmentary homology to several proteins. Using a prokaryotic expression system, pBad TOPO-47 cDNA, a 47-kDa recombinant protein was obtained and purified to apparent homogeneity by nickel-nitrilotriacetic acid resin and collagen affinity column. The recombinant protein binds to type III but not type I collagen-Sepharose 2B affinity columns. Anti-47-kDa but not anti-65-kDa antibody inhibits the binding of the recombinant protein to the type III collagen-coated micro titer wells in a dose-dependent manner. Like the receptor protein purified from platelet membranes, the recombinant protein inhibits type III collagen-induced platelet aggregation also in a dose-dependent manner. We have defined two active peptides from the cloned deduced amino acid sequence. Both peptides inhibit type III but not type I collagen-induced platelet aggregation in a dose-dependent fashion. These results suggest that the active binding site of the platelet receptor to type III collagen resides in these portions of the protein.     

Prevalence of HSP47 antigen and autoantibodies to HSP47 in the sera of patients with mixed connective tissue disease

The 47-kDa heat shock protein (HSP47) is an endoplasmic reticulum molecular chaperone that assists in the maturation of collagen molecules and whose expression is known to be upregulated in lesions of fibrotic diseases. We examined the levels of HSP47 protein and autoantibodies to HSP47 in the sera of patients with rheumatic autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, Sj?gren's syndrome, and mixed connective tissue disease (MCTD) by enzyme-linked immunosorbent assay and immunoblot analysis. Patients with idiopathic pulmonary fibrosis (IPF) were assessed as an example of non-autoimmune fibrotic disease. HSP47 antigen and autoantibody levels are significantly elevated in the sera of the rheumatic autoimmune disease patients, but not in the sera of the IPF patients. The sera of the MCTD patients showed particularly high levels of HSP47 antigen relative to healthy controls (1.99+/-0.22 vs 0.41+/-0.07 ng/ml). Autoantibodies to HSP47 were also in high levels in the sera of MCTD patients. These results suggest that simultaneous occurrence of systemic inflammation and upregulation of HSP47 caused leakage of HSP47 from fibrotic lesions into the peripheral blood, and the leaked antigen induced high titer of autoantibodies to HSP47. The high levels of HSP47 antigen and autoantibody may be useful blood markers of MCTD.     

The possible role of colligin/HSP47, a collagen-binding protein, in the pathogenesis of human and experimental fibrotic diseases.

Colligin or heat shock protein 47 (HSP47) is a stress protein that resides in the endoplasmic reticulum and is thought to participate in intracellular processing, folding, assembly and secretion of procollagens. Irrespective of the tissue site and organ, induction of colligin/HSP47 expression is always noted during the process of fibrosis, particularly in and around the fibrotic lesions in both humans and experimental models. Its expression is highly tissue- and cell-specific, and restricted to mostly phenotypically altered collagen-producing cells. These observations suggest that upregulation of this collagen-specific chaperone-colligin/HSP47 may play an important role in the subsequent fibrotic process, possibly by regulating increased synthesis/assembly of collagens.     

Specific recognition of the collagen triple helix by chaperone HSP47. II. The HSP47-binding structural motif in collagens and related proteins

The endoplasmic reticulum-resident chaperone heat-shock protein 47 (HSP47) plays an essential role in procollagen biosynthesis. The function of HSP47 relies on its specific interaction with correctly folded triple-helical regions comprised of Gly-Xaa-Yaa repeats, and Arg residues at Yaa positions have been shown to be important for this interaction. The amino acid at the Yaa position (Yaa(-3)) in the N-terminal-adjoining triplet containing the critical Arg (defined as Arg(0)) was also suggested to be directly recognized by HSP47 (Koide, T., Asada, S., Takahara, Y., Nishikawa, Y., Nagata, K., and Kitagawa, K. (2006) J. Biol. Chem. 281, 3432-3438). Based on this finding, we examined the relationship between the structure of Yaa(-3) and HSP47 binding using synthetic collagenous peptides. The results obtained indicated that the structure of Yaa(-3) determined the binding affinity for HSP47. Maximal binding was observed when Yaa(-3) was Thr. Moreover, the required relative spatial arrangement of these key residues in the triple helix was analyzed by taking advantage of heterotrimeric collagen-model peptides, each of which contains one Thr(-3) and one Arg(0). The results revealed that HSP47 recognizes the Yaa(-3) and Arg(0) residues only when they are on the same peptide strand. Taken together, the data obtained led us to define the HSP47-binding structural epitope in the collagen triple helix and also define the HSP47-binding motif in the primary structure. A motif search against human protein database predicted candidate clients for this molecular chaperone. The search result indicated that not all collagen family proteins require the chaperoning by HSP47.     

Sequence-specific recognition of collagen triple helices by the collagen-specific molecular chaperone HSP47

HSP47 is a molecular chaperone that plays an unknown role during the assembly and transport of procollagen. Our previous studies showed that, unlike most chaperones, HSP47 interacts with a correctly folded substrate. We suggested that HSP47 either stabilizes the correctly folded collagen helix from heat denaturation or prevents lateral aggregation prior to its transport from the endoplasmic reticulum. In this study we have addressed the role of triple helix stability in the binding of HSP47 to procollagen by expressing procollagen molecules with differing thermal stabilities and analyzing their ability to interact with HSP47 within the endoplasmic reticulum. Our results show that HSP47 interacts with thermostable procollagen molecules, suggesting that helix stabilization is not the primary function of HSP47 and that the interaction of HSP47 with procollagen depends upon the presence of a minimum of one Gly-X-Arg triplet within the triple helical domain. Interestingly, procollagen chains containing high proportions of stabilizing triplets formed triple helices and interacted with HSP47 even in the absence of proline hydroxylation, demonstrating that recognition does not depend upon this modification. Our results support the view that HSP47 functions early in the secretory pathway by preventing the lateral aggregation of procollagen chains.     

SPARC/SFN interaction, suppresses type I collagen in dermal fibroblasts

We previously suggested that keratinocyte releasable factors might modulate the wound healing process by regulating the expression of key extracellular matrix components such as collagenase (matrix metalloproteinase-1) and type I collagen in fibroblasts. The first one, we called it keratinocyte-derived anti-fibrogenic factor (KDAF), identified as stratifin (SFN) also named 14-3-3σ, revealing a strong collagenase activity. However, the second factor, which we named keratinocyte-derived collagen-inhibiting factor(s) (KD-CIF) that has shown to control the synthesis of type I collagen, was not known. Upon conducting a series of systematic protein purification methods followed by mass spectroscopy, two proteins: secreted protein acidic rich in cystein (SPARC) and SFN were identified in keratinocyte-conditioned media. Using co-immunoprecipitation and 3D modeling, we determined that SFN and SPARC form a complex thereby controlling the type I collagen synthesis and expression in fibroblasts. The levels of these proteins in fibrotic tissues (animal and human) were also evaluated and a differential expression of these proteins between normal and fibrotic tissue confirmed their potential role in development of fibrotic condition. In conclusion, this study describes for the first time an interaction between SPARC and SFN that may have implications for the regulation of matrix deposition and prevention of dermal fibrotic conditions such as hypertrophic scars and keloid.     

Quantitative assay of types I and III collagen synthesized by keloid biopsies and fibroblasts

Molecular sieve column chromatography was used to determine the amount of type I and III collagen synthesized by normal dermis and keloid biopsies and fibroblasts derived from these tissues. After incubation with radioactive proline, the collagen was extracted and separated into types I and III and then quantitated. There was no significant difference in the percent type III collagen synthesized by fresh keloid biopsies compared to normal dermis. Likewise, there was no significant difference in the percent type III collagen synthesized by keloid fibroblasts compared to normal dermal fibroblasts, However, fibroblasts from both keloid and normal dermis synthesized a lower percentage of type III collagen in cell culture compared to the original biopsies. These findings demonstrate that keloid collagen has the same type distribution as normal dermis and suggest that increased collagen synthesis in these lesions is not related to altered collagen types.     

Expression of constitutive and inducible HSP70 and HSP47 is enhanced in cells persistently spread on OPN1 or collagen

Cells persistently spread on OPN or collagen survive heat shock better than cells transiently spread on fibronectin or tissue culture plates. Thus, a central question is whether constitutively or inducible stress proteins are enhanced in cells grown on adhesive proteins that maintain a persistent spread cell shape. Levels of Hsp 72,73, and colligin/Hsp47 were determined by Western blot analyses. The inducible Hsp 72 was prominently expressed following heat shock in cells grown on OPN or collagen, but not in cells plated on fibronectin coated substratum or on tissue culture plates. Colligin/Hsp 47 and Hsp 73 manifested a similar pattern of expression indicating that these adhesive attachment proteins accommodate cell function through organization of cell architecture.     

In vivo glycosylation of dermal and tendon type I collagen

Recent studies show that native collagen fibers in the extracellular space can be subject to nonenzymatic glycosylation and that the extent of such glycosylation increases in clinical hyperglycemia and aging. In the present study, a comparison was made on the extent of glycosylation in rat tail tendon and in the soluble and insoluble fractions of collagen separated from rat skin after in vivo labeling with [14C]glucose. It was observed that nonenzymatic glycosylation occurred maximally in the salt-soluble fraction as measured by the level of ketoamine linked hexose. 14C radioactivity incorporation as well as the number of free amino groups was also increased in this fraction. However, the amounts of O-glycosidically linked sugars did not show much variation between the soluble and insoluble fractions. These findings could be correlated to the enhanced metabolic turnover of newly synthesized collagen in diabetics.     

Dermal collagen fibrils are hybrids of type I and type III collagen molecules

It has been suggested that dermal collagen fibrils with 67-nm periodicity consist of hybrids of type I and type III collagens. This is based on the assumption that all these banded fibrils are coated with type III collagen regardless of their diameter. However, conclusive evidence for this form of hybridization is lacking. In order to clarify this problem dermal collagen fibrils were disrupted into microfibrils using 8 M urea. Single and double indirect immunoelectron microscopy showed type III collagen at the periphery of intact collagen fibrils but no labeling with type I collagen antibodies, suggesting that the epitopes for this collagen were masked. Disrupted collagen fibrils revealed type I collagen throughout the fibril except for the periphery which was coated with type III collagen. Almost no type III collagen was noted in the interior of the collagen fibrils. Since type III collagen is present only at the periphery it suggests that this collagen has a different role than type I collagen and may have a regulatory function in fibrillogenesis.     

The change of HSP47, collagen specific molecular chaperone, expression in rat skeletal muscle may regulate collagen production with gravitational conditions.

It is well known that unloading of skeletal muscle with spaceflight leads skeletal muscle atrophy. However, it remains unclear how the extracellular matrix within the muscle and the connective tissues such as tendon and ligament respond to reduced mechanical load including microgravity, although they have been thought to play important roles in both the transmission of force and the signal transduction between cells and tissues. Type-I collagen and type-IV collagen, both of the major components of extracellular matrix and connective tissues. We focused on change of these collagen synthesis with mechanical load. To obtain an insight into the effects of gravitational changing on the protein metabolism of collagen in skeletal muscle during mechanical unloading, reloading after unloading, we investigated changes in the amount of Heat shock protein 47 (HSP47), has been postulated to be a collagen-specific molecular chaperone localized in the ER (Nagata et al, 1992). Western blot analysis revealed that HSP47 in rat soleus muscle decreases at 5 days after hindlimb suspension (HS). On the other hand, HSP47 in rat soleus muscle increases at 5 days after hypergravity (HG) induced by the centrifugation. RT-PCR analysis showed HSP47 mRNA decreased with HS earlier, as compared with collagen type-I and type-IV mRNA. From these results, the amount of HSP47 changing by gravitational condition may effect on signal transfers in the primary stage of adaptation and the change of HSP47 expression in skeletal muscle may regulate collagen production with gravitational conditions.     

HSP47 binds cooperatively to triple helical type I collagen but has little effect on the thermal stability or rate of refolding

HSP47, a collagen-specific molecular chaperone, interacts with unfolded and folded procollagens. Binding of chicken HSP47 to native bovine type I collagen was studied by fluorescence quenching and cooperative binding with a collagen concentration at half saturation (K(half)) of 1.4 x 10(-7) m, and a Hill coefficient of 4.3 was observed. Similar results are observed for the binding of mouse HSP47 recombinantly expressed in Escherichia coli. Chicken HSP47 binds equally well to native type II and type III procollagen without the carboxyl-terminal propeptide (pN type III collagen), but binding to triple helical collagen-like peptides is much weaker. Weak binding occurred to both hydroxylated and nonhydroxylated collagen-like peptides, and a significant chain length dependence was observed. Binding of HSP47 to native type I collagen had no effect on the thermal stability of the triple helix. Refolding of type I collagen in the presence of HSP47 showed minor changes, but these are probably not biologically significant. Binding of HSP47 to bovine pN type III collagen has only minor effects on the thermal stability of the triple helix and does not influence the refolding kinetics of the triple helix.     

Quantitative assay of types I and III collagen synthesized by keloid biopsyes and fibroblasts.

Molecular sieve column chromatography was used to determine the amount of type I and III collagen synthesized by normal dermis and keloid biopsies and fibroblasts derived from these tissues. After incubation with radioactive proline, the collagen was extracted and separated into types I and III and then quantitated. There was no significant difference in the percent type III collagen synthesized by fresh keloid biopsies compared to normal dermis. Likewise, there was no significant difference in the percent type III collagen synthesized by keloid fibroblasts compared to normal dermal fibroblasts. However, fibroblasts from both keloid and normal dermis synthesized a lower percentage of type III collagen in cell culture compared to the original biopsies. These findings demonstrate that keloid collagen has the same type distribution as normal dermis and suggest that increased collagen synthesis in these lesions is not related to altered collagen types.     

Human bone contains type III collagen, type VI collagen, and fibrillin: type III collagen is present on specific fibers that may mediate attachment of tendons, ligaments, and periosteum to calcified bone cortex

We evaluated the distribution of Type III collagen, Type VI collagen, and fibrillin in human bone, using monoclonal antibodies (MAb) of proven specificity. All three molecules are present in developing and remodeling bone. Type III collagen is present in discrete fiber bundles throughout the bone cortex but is concentrated at the Haversian canal surface and in the fibers at the bone-periosteal interface. The collagen fibrils in these bundles are of uniform diameter. Type III-containing collagen fibers are detected at all ages examined, from 30 fetal weeks to 80 years. Type VI collagen is present in fetal bone in discrete fibrils separate from Type III collagen, and becomes restricted to the margins of bone cells and the bone surface by 7 years. The distribution of fibrillin resembles that of Type III collagen in the fetus, but at 7 years is absent from the interior of the cortex except for the canaliculi and cement lines, and remains concentrated in discrete fibers at the bone surface.     

Xaa-Arg-Gly triplets in the collagen triple helix are dominant binding sites for the molecular chaperone HSP47.

HSP47 is an essential procollagen-specific molecular chaperone that resides in the endoplasmic reticulum of procollagen-producing cells. Recent advances have revealed that HSP47 recognizes the (Pro-Pro-Gly)(n) sequence but not (Pro-Hyp-Gly)(n) and that HSP47 recognizes the triple-helical conformation. In this study, to better understand the substrate recognition by HSP47, we synthesized various collagen model peptides and examined their interaction with HSP47 in vitro. We found that the Pro-Arg-Gly triplet forms an HSP47-binding site. The HSP47 binding was observed only when Arg residues were incorporated in the Yaa positions of the Xaa-Yaa-Gly triplets. Amino acids in the Xaa position did not largely affect the interaction. The recognition of the Arg residue by HSP47 was specific to its side-chain structure because replacement of the Arg residue by other basic amino acids decreased the affinity to HSP47. The significance of Arg residues in HSP47 binding was further confirmed by using residue-specific chemical modification of types I and III collagen. Our results demonstrate that Xaa-Arg-Gly sequences in the triple-helical procollagen molecule are dominant binding sites for HSP47 and enable us to predict HSP47-binding sites in homotrimeric procollagen molecules.     

Characterization of a novel transformation-sensitive heat-shock protein (HSP47) that binds to collagen

The synthesis of a major collagen-binding glycoprotein of molecular weight 47,000 was previously shown to be regulated by malignant transformation as well as by heat shock in chick embryo fibroblasts. The 47-kDa protein purified from chick embryos was characterized biochemically, and was found to exist as a monomer in native form. Its composition was enriched in basic amino acids and glycine, with fewer acidic residues and virtually no cysteine. N-terminal amino acid sequencing covering 36 residues revealed a single, novel sequence with an internal tandem repeat of Asp-Lys-Ala-Thr-Thr-Leu-Ala and Asp-Arg-Ser-Thr-Thr-Leu-Ala.