A collagen-binding glycoprotein from bovine platelets is identical to propolypeptide of von Willebrand factor

Several proteins from bovine platelet lysate bound to type I collagen immobilized to the beads of formyl derivatives of cellulose. Among these proteins, a protein of about 100,000 daltons was purified to homogeneity by two additional affinity chromatographies, an organomercurial-agarose and a lentil lectin-agarose. This protein consisted of a single polypeptide chain which contains carbohydrate moiety and many intrapolypeptide disulfide bridges. In addition to platelets, this protein was present in plasma and cultured endothelial cells but not in red blood cells, leukocytes, and smooth muscle cells. Furthermore, it was released from platelets upon stimulation by various agonists. The purified 100-kDa protein was labeled with 125I to quantitate its binding to fibrillar type I collagen. The protein specifically bound to fibrillar collagen with the apparent dissociation constant of 5.6 x 10(-8) M for the high affinity site and 5.5 x 10(-7) M for the low affinity site. Analyses of amino acid sequences of both intact and tryptic fragments of this protein revealed that it had strong homology to the propolypeptide of human von Willebrand factor, which is also known as von Willebrand antigen II. Various properties of this protein listed above also strongly suggest that it was indeed the propolypeptide of bovine von Willebrand factor.     

Stimulation of pro-MMP-2 production and activation by native form of extracellular type I collagen in cultured hepatic stellate cells

Cultured hepatic stellate cells (HSCs) are known to change their morphology and function with respect to the production of extracellular matrices (ECMs) and matrix metalloproteinases (MMPs) in response to ECM components. We examined the regulatory role of the native form of type I collagen fibrils in pro-MMP-2 production and activation in cultured HSCs. Gelatin zymography of the conditioned media revealed that pro- and active form of MMP-2 was increased in the HSCs cultured on type I collagen gel but not on type I collagen-coated surface, gelatin-coated surface, type IV collagen-coated surface, or Matrigel, suggesting the importance of the native form of type I collagen fibrils in pro-MMP-2 production and activation. The induction of active MMP-2 by extracellular type I collagen was suppressed by the blocking antibody against integrin beta1 subunits, indicating the involvement of integrin signaling in pro-MMP-2 activation. RT-PCR analysis indicated that MMP-2, membrane type-1 MMP (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA levels were elevated in HSCs cultured on type I collagen gel. The increased MT1-MMP proteins were localized on the cell surface of HSCs cultured on type I collagen gel. In contrast to the expression of MMP-2, HSCs showed a great decline in MMP-13 expression in HSCs cultured on type I collagen gel. These results indicate that the native fibrillar (polymerized) but not monomeric form of type I collagen induced pro-MMP-2 production and activation through MT1-MMP and TIMP-2 in cultured HSCs, suggesting an important role of HSCs in ECM remodeling in the hepatic perisinusoidal spaces.     

Soluble collagen VI drives serum-starved fibroblasts through S phase and prevents apoptosis via down-regulation of Bax

We previously showed that soluble, pepsin-solubilized collagen VI increases de novo DNA synthesis in serum-starved HT1080 and 3T3 fibroblasts up to 100-fold compared with soluble collagen I, reaching 80% of the stimulation caused by 10% fetal calf serum. Here we show that collagen VI also inhibits apoptotic cell death in serum-starved cells as evidenced by morphological criteria, DNA laddering, complementary apoptosis assays (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, enzyme-linked immunosorbent assay, and fluorescence-activated cell sorting), and quantification of apoptosis-regulating proteins. In the presence of starving medium alone or collagen I, the proapoptotic Bax was up-regulated 2-2.5-fold, compared with soluble collagen VI and fetal calf serum, whereas levels of the antiapoptotic Bcl-2 protein remained unaffected. In accordance with its potent stimulation of DNA synthesis, soluble collagen VI carries serum-starved HT1080 and Balb 3T3 fibroblasts through G(2) as shown by fluorescence-activated cell sorting analysis, whereas cells exposed to medium and collagen I where arrested at G(1)-S. This was accompanied by a 2-3-fold increase in cyclin A, B, and D1 protein expression. Collagen VI-induced inhibition of apoptotic cell death may be operative during embryogenesis, wound healing, and fibrosis when elevated tissue and blood levels of collagen VI are observed, thus initiating a feedback loop of mesenchymal cell activation and proliferation.     

Effect of forskolin on collagen production in clonal osteoblastic MC3T3-E1 cells

The effect of forskolin on collagen production in osteoblasts was investigated by using clonal osteoblastic MC3T3-E1 cells cultured in a-minimum essential medium containing 0.1% bovine serum albumin. Forskolin increased the adenylate cyclase activity in membranes pelleted from homogenates of the cell line in a dose-dependent manner. The drug caused a 13-fold stimulation at 10(-4) M, indicating that the compound directly acts on adenylate cyclase, leading to an increase in the intracellular cAMP content of the cells. Collagen accumulation in the cultures was elevated by one-day treatment with 5 X 10(-5) M forskolin to about twice that in the controls. The stimulation was mainly due to an elevation in collagen synthesis but not to an inhibition of intracellular collagen degradation because forskolin dose-dependently increased collagen synthesis; it also significantly increased the amount of low-molecular-weight hydroxyproline found in the cultures. Cells treated with forskolin produced mainly type I collagen, as found in bone matrix in situ, with only small amounts of other types of collagen. Furthermore, forskolin time-dependently inhibited DNA synthesis in the cells, indicating that the increase in type I collagen synthesis by forskolin was not due to stimulated cell proliferation. These results suggest that cAMP is closely linked to the differentiation of osteoblasts in vitro.     

Involvement of the MAP kinase pathways in induction of GADD45 following UV radiation

Indian hedgehog (Ihh) is highly expressed in prehypertrophic chondrocytes in vivo and has been proposed to regulate the proliferation and maturation of chondrocytes and bone collar formation in the growth plate. In high-density cultures of rabbit growth-plate chondrocytes, Ihh mRNA was also expressed at the highest level in the prehypertrophic stage. To explore endogenous factors that regulate Ihh expression in chondrocytes, we examined the effects of various growth factors on Ihh mRNA expression in this system. Retinoic acid (RA) and bone morphogenetic protein-2 enhanced Ihh mRNA expression, whereas PTH/PTH-related peptide (PTHrP) markedly suppressed Ihh expression. RA at more than 10(-8) M induced the expression of Ihh and Patched 1 (Ptc1) within 3 h, before it increased the type X collagen mRNA level at 6-24 h. Cycloheximide blocked the up-regulation of Ihh by RA, indicating the requirement of de novo protein synthesis for this stimulation. These findings suggest that RA is involved in the up-regulation of Ihh during endochondral bone formation. In contrast to RA, PTH (1-84) at 10(-7) M abolished the mRNA expression of Ihh and Ptc1 within 2-4 h, before it suppressed the expression of type X collagen at 12-24 h. The inhibition of Ihh expression by PTH (1-84) did not require de novo protein synthesis. PTH (1-34), PTHrP (1-34), and (Bu)(2)cAMP also suppressed Ihh expression. On the other hand, Ihh has been reported to induce PTHrP synthesis in the perichondrium. Consequently, the direct inhibitory action of PTH/PTHrP on Ihh appears to be a negative feedback mechanism that prevents excess PTHrP accumulation in cartilage.     

Collagen synthesis as a marker for cell type in mouse 3T3 lines

Collagen synthesis was applied as a test of the fibroblastic or endothelial origin of the 3T3 mouse lines. Swiss/3T3 lines and Balb/3T3 (clone A31) were compared with respect to the amounts and types of collagen synthesized. All lines symthesized collagen at relatively high and similar rates. For all lines, 75-90% of the collagen synthesized was type I, and the remainder was type III. There was no evidence for synthesis of type IV collagen. By all these parameters, Swiss and Balb/3T3 lines resemble cultured fibroblasts rather than cultured vascular endothelia.     

Role of TGF-beta1 in relation to exercise-induced type I collagen synthesis in human tendinous tissue.

Mechanical loading of tissue is known to influence local collagen synthesis, and microdialysis studies indicate that mechanical loading of human tendon during exercise elevates tendinous type I collagen production. Transforming growth factor-beta1 (TGF-beta1), a potent stimulator of type I collagen synthesis, is released from cultured tendon fibroblasts in response to mechanical loading. Thus TGF-beta1 could link mechanical loading and collagen synthesis in tendon tissue in vivo. Tissue levels of TGF-beta1 and type I collagen metabolism markers [procollagen I COOH-terminal propeptide (PICP) and COOH-terminal telopeptide of type I collagen (ICTP)] were measured by microdialysis in peritendinous tissue of the Achilles' tendon in six male volunteers before and after treadmill running (1 h, 12 km/h, 3% uphill). In addition, blood levels of TGF-beta1, PICP, and ICTP were obtained. PICP levels increased 68 h after exercise (P < 0.05). Dialysate levels of TGF-beta1 changed from 303 +/- 46 pg/ml (at rest) to 423 +/- 86 pg/ml 3 h postexercise. This change was nonsignificant, but the decay of tissue TGF-beta1 after catheter insertion was markedly delayed by exercise compared with the decay seen in resting subjects. Plasma concentrations of TGF-beta1 rose 30% in response to exercise (P < 0.05 vs. pre). Our observations indicate an increased local production of type I collagen in human peritendinous tissue in response to uphill running. Although not conclusive, changes in circulating and local TGF-beta1, in response to exercise, suggest a role for TGF-beta1 in mechanical regulation of local collagen type I synthesis in tendon-related connective tissue in vivo.     

Selective adhesion of macrophages to denatured forms of type I collagen is mediated by scavenger receptors.

Macrophages (Mφs) are multifunctional immune cells which are involved in the regulation of immune and inflammatory responses, as well as in tissue repair and remodeling. In tissues, Mφs reside in areas which are rich in extracellular matrix (ECM), the structural component which also plays an essential role in regulating a variety of cellular functions. A major ECM protein encountered by Mφs is type I collagen, the most abundant of the fibril-forming collagens. In this study, the adhesion of RAW 264.7 murine Mphis to native fibrillar, monomeric, and denatured type I collagen was investigated. Using atomic force microscopy, structural differences between fibrillar and monomeric type I collagen were clearly resolved. When cultured on fibrillar type I collagen, Mphis adhered poorly. In contrast, they adhered significantly to monomeric, heat-denatured, or collagenase-modified type I collagen. Studies utilizing anti-beta1 and -beta2 integrin adhesion-blocking antibodies, RGD-containing peptides, or divalent cation-free conditions did not inhibit Mphi; adhesion to monomeric or denatured type I collagen. However, macrophage scavenger receptor (MSR) ligands and anti-MSR antibodies significantly blocked Mphi; adhesion to denatured and monomeric type I collagen strongly suggesting the involvement of the MSR as an adhesion molecule for denatured type I collagen. Further analysis by Western blot identified the MSR as the primary receptor for denatured type I collagen among Mphi; proteins purified from a heat-denatured type I collagen affinity column. These findings indicate that Mphis adhere selectively to denatured forms of type I collagen, but not the native fibrillar conformation, via their scavenger receptors.     

Cartilage proteoglycan aggregate and fibronectin can modulate the expression of type X collagen by embryonic chick chondrocytes cultured in collagen gels

Chick embryo sternal chondrocytes from the caudal and cephalic regions were cultured within type I collagen gels and type I collagen/proteoglycan aggregate composite gels in normal serum. Caudal region chondrocytes were also cultured within type I collagen gels in the presence of fibronectindepleted serum. There was a marked stimulation of type X collagen synthesis by the caudal region chondrocytes after 9 days in the presence of fibronectin-depleted serum and after 14 days in the presence of proteoglycan aggregate. These results provide evidence for the ability of chondrocytes from a zone of permanent cartilage to synthesise type X collagen and for the involvement of extracellular matrix components in the control of type X collagen gene expression.     

Collagen biosynthesis in cultured rat testicular Sertoli and peritubular myoid cells.

The incorporation of 3H-proline into protein was regarded as a measure of total protein synthesis and the incorporation into hydroxyproline as indicative of collagen synthesis. Relative collagen synthesis (expressed as percent of total protein synthesized) by Sertoli and peritubular myoid cells cultured from 20-22 day old rat testis was estimated. In both secreted and cellular pools, relative collagen synthesis by Sertoli cells was significantly greater than by peritubular myoid cells. Coculture of Sertoli and myoid cells resulted in a significant increase in relative collagen synthesis when compared to monocultures of each cell type. Addition of serum to peritubular myoid cells resulted in a stronger stimulation of relative collagen production. Sertoli cell extracellular matrix inhibited relative collagen synthesis by peritubular myoid cells in the presence or absence of serum. Radioactivity into hydroxyproline as corrected per cellular DNA also showed similar results. Immunolocalization studies confirmed that both cell types synthesize type I and type IV collagens. These results indicate that stimulation of collagen synthesis observed in Sertoli-myoid cell cocultures is due to humoral interactions, rather than extracellular matrix, and Sertoli cell extracellular matrix regulates serum-induced increase in collagen synthesis by peritubular myoid cells.     

The effects of collagen synthesis inhibitory drugs on somitogenesis and myogenin expression in cultured chick and mouse embryos

The role of fibrillar collagen on myogenic differentiation has previously been studied in tissue culture cell lines but has not been studied in situ. We treated cultured chick and mouse embryos with collagen synthesis inhibitors to determine the role of fibrillar collagen on somitogenesis and on myogenic differentiation in vivo. Stage 12 chick embryos and 8.7 dpc mouse embryos were cultured in control medium or a range of concentrations of the collagen synthesis inhibitors ethyl-3,4-dihydroxybenzoate (EDHB) or cis-hydroxy-proline (CHP). Chick embryos were cultured for 24 h and mouse embryos were cultured for 30 h. Both collagen synthesis inhibitors produced a range of somite abnormalities including formation of fewer and irregular somites in both chick and mouse at high drug concentrations, as well as formation of double somites in EDHB-treated chick embryos. Examination of EDHB-treated mouse embryos by scanning electron microscopy demonstrated a dosage-dependent loss of fibrillar collagen and associated extracellular matrix. Expression of myogenin in EDHB-treated mouse embryos, examined by whole-mount in situ hybridization, was suppressed at higher dosage levels. This study suggests that inhibition of fibrillar collagen production and/or loss of fibrillar collagen in the developing avian and mammalian embryo results in abnormal somite formation and perturbed myogenic differentiation.     

Deposition and ultrastructural organization of collagen and proteoglycans in the extracellular matrix of gel-cultured fibroblasts

Human skin fibroblasts were cultivated within the three-dimensional space of polymerized alginate and collagen, respectively. The in vitro synthesis of collagens and proteoglycans was measured during the first 3 days of culture, and the deposition as well as the ultrastructural organization of newly synthesized extracellular matrix components were examined by electron microscopy. The amount of collagens and proteoglycans synthesized by fibroblasts, embedded in calcium alginate gels as well as in collagen lattices, was lowered as compared to monolayer cultures. Furthermore, it was found that collagen synthesis was reduced to a greater extent in alginate gels than in collagen lattices. On the contrary, total proteoglycan biosynthesis was similarly reduced either in alginate gels or in collagen lattices. At the end of a 3-day-culture period, filamentous material as well as cross-striated banded structures were found extracellularly in the alginate gel. According to their periodicity, their banding pattern, their association with polyanionic matrix components and their sensitivity towards glycosaminoglycan-degrading enzymes we could distinguish (1) sheets of amorphous non-banded material consisting of irregularly arranged filaments and containing dermatan sulfate-rich proteoglycans (type I structures), (2) sheets of long-spacing fibrils consisting of parallel orientated filaments and containing chondroitin sulfate-rich proteoglycans (= zebra bodies; type II structures), and (3) fibrillar structures with a complex banding pattern different from that of native collagen fibrils (type III structures). In fibroblasts cultured in collagen lattices, we only sporadically found depositions which are identified as type I structures. Using indirect immunoelectron microscopy and monospecific polyclonal antibodies, we localized type VI collagen in type I structures and type II structures. Type III structures can be identified as type I collagen derived as becomes obvious by comparison with segment long spacing crystallites of type I collagen.     

Expression of extracellular matrix components is regulated by substratum

Reconstituted basement membranes and extracellular matrices have been demonstrated to affect, positively and dramatically, the production of milk proteins in cultured mammary epithelial cells. Here we show that both the expression and the deposition of extracellular matrix components themselves are regulated by substratum. The steady-state levels of the laminin, type IV collagen, and fibronectin mRNAs in mammary epithelial cells cultured on plastic dishes and on type I collagen gels have been examined, as has the ability of these cells to synthesize, secrete, and deposit laminin and other, extracellular matrix proteins. We demonstrate de novo synthesis of a basement membrane by cells cultured on type I collagen gels which have been floated into the medium. Expression of the mRNA and proteins of basement membranes, however, are quite low in these cultures. In contrast, the levels of laminin, type IV collagen, and fibronectin mRNAs are highest in cells cultured on plastic surfaces, where no basement membrane is deposited. It is suggested that the interaction between epithelial cells and both basement membrane and stromally derived matrices exerts a negative influence on the expression of mRNA for extracellular matrix components. In addition, we show that the capacity for lactational differentiation correlates with conditions that favor the deposition of a continuous basement membrane, and argue that the interaction between specialized epithelial cells and stroma enables them to create their own microenvironment for accurate signal transduction and phenotypic function.     

Mechanisms of stimulation of collagen synthesis during chick embryonic skin development.

To study how collagen synthesis is regulated in developing chick embryonic skin, hydroxyproline synthesis, incorporation of proline, and translational activity and content of collagen mRNA in 12-, 15-, and 18-day skins were determined and compared with each other. Hydroxyproline synthesis in the 18-day skins was markedly increased over that in the 12-day skins, whereas proline incorporation was moderately increased. The increase in collagen synthesis from day 15 to 18 was accompanied by increases in both the translational activity and the content of type I procollagen mRNA, with a selective increase in the lower-molecular-weight species of pro alpha 1 (I) collagen mRNA. In contrast, the stimulation of collagen synthesis from day 12 to day 15 did not parallel the levels of type I procollagen mRNA. These results suggest that the stimulation of collagen synthesis is regulated by collagen mRNA levels only in the later stage of development (from day 15 to day 18). Both the collagen synthesis and type I procollagen mRNA levels in the fibroblasts isolated on each corresponding day were constant. The difference in collagen synthesis under two different culture conditions suggests that cell-matrix interaction and/or some serum factors, including several growth factors, are essential for the marked stimulation of collagen synthesis observed in 12- to 18-day skin.