Subcellular localization of procollagen I and prolyl 4-hydroxylase in corneal endothelial cells

To investigate the molecular mechanism of intracellular degradation of type I collagen in normal corneal endothelial cells (CEC), we studied the role of prolyl 4-hydroxylase (P4-H) and protein disulfide-isomerase (PDI; the beta subunit of P4-H) during procollagen I biosynthesis. When the subcellular localization of P4-H and PDI was determined, P4-H demonstrated a characteristic diffuse endoplasmic reticulum (ER) pattern, whereas PDI showed a slightly more restricted distribution within the ER. When colocalization of procollagen I with the enzymes was examined, procollagen I and PDI showed a large degree of colocalization. P4-H and procollagen I were predominantly colocalized at the perinuclear site. When colocalization of type IV collagen with PDI and P4-H was examined, type IV collagen was largely colocalized with PDI, which showed a wider distribution than type IV collagen. Type IV collagen is similarly colocalized with P4-H, except in some perinuclear sites. The colocalization profiles of procollagen I with both PDI and P4-H were not altered in cells treated with alpha,alpha'-dipyridyl compared to those of the untreated cells. The underhydroxylated type IV collagen demonstrated a colocalization profile with PDI similar to that observed with procollagen I, while the underhydroxylated type IV collagen was predominantly colocalized with P4-H at the perinuclear sites. Immunoblot analysis showed no real differences in the amounts of the beta subunit/PDI and the catalytic alpha subunit of P4-H in CEC compared to those of corneal stromal fibroblasts (CSF). When protein-protein association was determined, procollagen I was associated with PDI much more in CEC than it was in CSF, whereas type IV collagen showed no differential association specificity to PDI in both cells. Limited proteolysis of the newly synthesized intracellular procollagen I with pepsin showed that procollagen I in CEC was degraded by pepsin, whereas CSF contained type I collagen composed of alpha1(I) and alpha2(I). These findings suggest that procollagen I synthesized in CEC is not in triple helical conformation and that the improperly folded procollagen I may be preferentially associated with PDI before targeting to the intracellular degradation.     

A factor from damaged rat kidney stimulates collagen biosynthesis by mesangial cells

Rats were administered CCl4, a well-defined nephrotoxin, for 20 weeks to produce glomerular sclerosis. Tubular degeneration and necrosis with interstitial fibrosis was clearly evident by histological examination. Kidneys were homogenized in phosphate-buffered saline and a collagen synthesis-stimulating factor was isolated by Sephadex G-50 gel filtration. The 5 kDa component stimulated both type I and type IV procollagen synthesis by mesangial cells and type I procollagen synthesis by rat skin fibroblasts. In each cell type, 2-6-fold increases in procollagen protein production or cell proliferation was noted. The steady-state levels of mRNA encoding for procollagen alpha 1(I) and procollagen alpha 1(IV) chains in mesangial cells were determined by by hybridization to their corresponding cDNA clones. The type I procollagen mRNA was elevated 1.4-fold compared to a 1.6-fold increase in mRNA encoding for type IV procollagen. The similar properties and chemical characteristics of this fibrogenic factor with a factor from fibrotic liver suggests they are the same and that a common endogenous collagen synthesis stimulator may be present in fibrosing organs, thus providing a driving force for collagen over-production.     

Synthesis of [pro alpha 1(IV)]3 collagen molecules by cultured embryo-derived parietal yolk sac cells

Electron immunohistochemical studies demonstrate that cultured embryo-derived parietal yolk sac (ED-PYS) carcinoma cells synthesize type IV collagen. This material has been isolated and characterized. The collagen obtained after limited pepsin digestion from the medium in which the cells are grown is composed of homogeneous components with a molecular mass of approximately 95 000 daltons. When chromatographed on (carboxymethyl)cellulose under denaturing conditions, the chains elute as acidic components slightly before the human alpha 1(I) chain and coincident with the position of elution of the pepsin-derived human alpha 1(IV) chain. This analysis indicates the presence of a single type of collagen chain in the pepsin-derived ED-PYS synthesized material. In addition, the profile of cyanogen bromide (CNBr) cleavage products obtained from the pepsin-derived ED-PYS cell collagen chains is essentially identical with that derived from the human alpha 1(IV) chain. Isolation of the medium collagen in the absence of pepsin digestion reveals the presence of two high molecular weight components equivalent in size to procollagen alpha chains. However, both high molecular weight products yield CNBr cleavage products that correspond to those obtained from the pepsin-derived alpha 1(IV) chain. The ED-PYS cell-associated collagens obtained with or without the use of pepsin contain components that are essentially identical with those isolated from the culture-medium collagen. These data provide definitive evidence for the existence of type IV collagen molecules composed solely of alpha 1(IV) procollagen chains and further document the usefulness of ED-PYS cells for investigating the biosynthesis of basement membrane components.     

Biosynthesis and in vitro translation of type IV procollagens

The present paper describes how epithelial cells, cultured from bovine anterior lens capsule explants, synthesize and secrete procollagen type IV polypeptide chains alpha 1(IV) and alpha 2(IV). Metabolic labeling of these cells with [14C]proline for different time intervals and subsequent analysis by SDS/polyacrylamide gel electrophoresis revealed the presence of two polypeptide chains with apparent molecular masses of 180 kDa and 170 kDa. The procollagens were bacterial-collagenase-sensitive and were specifically immunoprecipitated by antibodies raised against the 7S domain of type IV collagen. Type IV procollagen poly(A)-rich RNA was isolated from cultured lens capsule cells and translated in a reticulocyte lysate cell-free system. Two polypeptides with apparent molecular masses of 152 kDa and 145 kDa were identified as procollagen type IV unmodified chains by gel electrophoresis, collagenase digestion and specific immunoprecipitation. During experiments in which cells were labeled in the presence of alpha, alpha'-bipyridyl, type IV procollagen appeared as one major band comigrating with a 145 kDa polypeptide on SDS-gel electrophoresis.     

Effects of endogenous carbon monoxide on collagen synthesis in pulmonary artery in rats under hypoxia

To study the role of endogenous carbon monoxide (CO) in collagen metabolism during hypoxic pulmonary vascular remodeling, a total of 18 Wistar rats were used in the study and they were randomly divided into three groups: hypoxia group (n = 6), hypoxia with zinc protoporphyrin-IX (ZnPP-IX) group (n = 6) and control group (n = 6). The measurement of mean pulmonary artery pressure (mPAP) and carboxyhemoglobin (HbCO) formation in lung tissue homogenates was measured. A morphometric analysis of pulmonary vessels was performed, in which the percentage of muscularized arteries (MA); partially muscularized arteries (PMA) and nonmuscularized arteries (NMV) in small and median pulmonary vessels, relative medial thickness (RMT) and relative medial area (RMA) of pulmonary arteries were analyzed. Collagen type I and III and transforming growth factor-beta3 (TGF-beta3) expressions were detected by immunohistochemical assay. The expressions of procollagen type I and III and TGF-beta3 mRNA were detected by in situ hybridization. The results showed that ZnPP-IX significantly increased mPAP and markedly decreased HbCO formation in lung tissue homogenates in rats under hypoxia (P < 0.01). In the hypoxia rats treated with ZnPP-IX, the percentage of muscularized arteries of small and median pulmonary vessels was obviously increased, and RMT and RMA of intra-acinar muscularized pulmonary arteries were markedly increased compared with hypoxic rats. Ultrastructural changes, such as hyperplasia and hypertrophy of endothelial cells (ECs) and smooth muscle cells (SMCs) and the increased number of SMCs in synthetic phenotype were found in intra-acinar pulmonary muscularized arteries of hypoxic rats treated with ZnPP-IX. Meanwhile, ZnPP-IX promoted the expression of collagen type I and III and TGF-beta3 protein in pulmonary arteries of rats under hypoxia (P < 0.01). Furthermore, ZnPP-IX elevated obviously the expressions of procollagen type I and III mRNA, and TGF-beta3 mRNA in pulmonary arteries of rats under hypoxia (P < 0.01). The results of this study suggested that ZnPP-IX played an important role in promoting collagen synthesis in pulmonary arteries of rats with hypoxic pulmonary structural remodeling by increasing the expression of TGF-beta3. The above findings also suggested a possible role of endogenous CO in the pathogenesis of chronic hypoxic pulmonary hypertension.     

Extracellular matrix proteins of human epidermal keratinocytes and feeder 3T3 cells

Cultures of human epidermal keratinocytes obtained from adult epidermis were initiated using irradiated BALB/3T3 cells as feeder layers. At different stages of confluence of the epidermal islands, feeder cells were removed and the extracellular matrix proteins of both pure component cells and cocultures were analyzed biochemically and by immunochemical methods and compared to those of skin fibroblasts of the same donors. The keratinocytes synthesized and secreted fibronectin and small amounts of laminin and type IV collagen. In addition, a nondisulfide-linked collagenous polypeptide (Mr = 120,000) was synthesized by the keratinocytes and was confined to the cell layers. Collagenous polypeptides with Mr = 120,000 were also synthesized by organ cultures of epidermal tissue and were detected in its acid or detergent extracts but again no secretion to culture medium was found. The Mr = 120,000 collagen had biochemical and immunological properties distinct from those of types I-V collagens. In immunofluorescence of keratinocyte cultures, fibronectin staining was prominent in the lining marginal cells of the expanding periphery of the epidermal cell islands but was not detected in the terminally differentiating cells in the upper layers of stratified colonies. Very little type IV collagen was found deposited in pericellular matrix form by the keratinocytes. In contrast, the mouse 3T3 feeder cells were found to produce both type IV collagen and laminin in addition to the previously identified connective tissue glycoproteins of fibroblasts, interstitial procollagens, and fibronectin. Basement membrane collagen of the 3T3 cells was found deposited as apparently unprocessed procollagen alpha 1(IV) and alpha 2(IV) chains. The production in culture conditions of basal lamina glycoproteins by the fibroblastic feeder cells may promote the attachment and growth of the cocultured keratinocytes.     

Elevated Cell Invasion Is Induced by Hypoxia in a Human Pituitary Adenoma Cell Line

Pituitary adenoma tissues are hypovascular, and have a lower partial oxygen pressure compared with neighboring normal organs. In this study, we investigated whether hypoxia influences the cell invasiveness of the human pituitary adenoma cell line, HP-75. HP-75 cells were exposed to hypoxic (1–10% oxygen) or normoxic (21% oxygen) conditions for 24 hours. Gelatin and reverse zymogram assays were used to determine the enzyme activities of matrix metalloproteinases (MMP) and tissue inhibitor of metalloproteinases (TIMP). Cell adhesion and Matrigel cell invasion were examined with a Boiden chamber. Finally, the mRNA gene expression profiles of cells exposed to hypoxia or normoxia were examined by cDNA microarray and confirmed with real-time RT-PCR and flow cytometry. The gelatin and reverse zymograms revealed that the activities of MMP and TIMP were not significantly altered by hypoxia. Matrigel cell invasion and cell adhesion to Matrigel or collagen type IV were increased by hypoxia (3.8- and 4.8-fold, respectively). The cDNA microarray analysis revealed that laminin β2 chain mRNA was specifically up-regulated under hypoxic conditions (4.96-fold). Finally, real-time RT-PCR and flow cytometry verified the elevated expression of laminin β2 chain at the mRNA and protein levels under hypoxic conditions. RNA interference with siRNA targeting laminin β2 inhibited Matrigel invasion and adhesion to collagen type IV in a dose.dependent manner.Collectively, these results suggested that hypoxia (1% oxygen) enhanced the cell invasion properties of a pituitary adenoma cell line in association with elevated expression of laminin β2 and enhanced binding to collagen type IV.Key Words: cell invasion, hypoxia, laminin β2, pituitary adenoma, siRNA     

Secretion of non-helical collagenous polypeptides of alpha1(IV) and alpha2(IV) chains upon depletion of ascorbate by cultured human cells

Our previous report showed that human fetal lung fibroblasts secreted non-disulfide-bonded, non-helical collagenous polypeptides of alpha1(IV) and alpha2(IV) chains depending on culture conditions [Connective Tissue (1999) 31, 161-168]. The secretion of non-helical collagenous polypeptides is unexpected from the current consensus that such polypeptides are not secreted under physiological conditions. The absence of interchain disulfide bonds among alpha1(IV) and alpha2(IV) chains was always correlated with the absence of triple-helical structure of the type IV collagen. The finding corresponds with the fact that the interchain disulfide bonds are formed at or close to the completion of the type IV collagen triple-helix formation. The present report shows that ascorbate is the primary factor for the triple-helix formation of the type IV collagen. When human mesangial cells were cultured with ascorbate, only the triple-helical type IV collagen was secreted. However, when the cells were cultured without ascorbate, the non-helical alpha1(IV) and alpha2(IV) chains were secreted. Relative amounts of the secreted products were unchanged with or without ascorbate, suggesting that ascorbate is required for the step of the triple-helix formation. The ascorbate-dependency of the triple-helix formation of the type IV collagen was observed in all the human cells examined. The non-helical alpha1(IV) chain produced by the ascorbate-free culture contained about 80% less hydroxyproline than the alpha1(IV) chain from the triple-helical type IV collagen. The evidence for the non-association of the non-helical alpha1(IV) and alpha2(IV) chains in the conditioned medium was obtained by an anti-alpha1(IV) antibody-coupled affinity column chromatography for the conditioned medium. Although all the non-helical alpha1(IV) chains were found in the bound fraction, all the non-helical alpha2(IV) chains were recovered in the flow-through fraction. The present findings suggest that ascorbate plays a key role in the trimerization step of three alpha chains and/or in the subsequent triple-helix formation of the type IV collagen.     

Hypoxia induces differential expression of the integrin receptors alpha(vbeta3) and alpha(vbeta5) in cultured human endothelial cells

The integrins alpha(vbeta3) and alpha(vbeta5) have been implicated in playing a key role in the process of angiogenesis. In this study, we examined the effects of hypoxia, an important stimulus of angiogenesis, on the differential expression of the integrin subunits beta(3) and beta(5). beta(3) and beta(5) messenger RNA (mRNA), protein levels, and alpha(v)beta(3) function were measured in human umbilical vein endothelial cells (HUVECs) cultured under normoxic and hypoxic (1% O(2)) conditions. Cells exposed to hypoxic conditions for up to 72 h showed gradually increased mRNA levels of alpha(V) and beta(3), peaking at 24 h, in comparison with cells cultured under normoxic conditions. However, beta(5) mRNA levels, under the same hypoxic conditions, remained at a constant level. Results from Western blot analysis of HUVECs, cultured under hypoxic conditions, paralleled those of the Northern analysis with an increased expression in alpha(v)beta(3) protein levels, measured by blotting with LM609, evident by 24 h. alpha(v)beta(5) protein levels, measured by blotting with P1F6, did not change for up to 72 h. HUVECs cultured under hypoxic conditions for 72 h showed increased attachment to fibrinogen, an alpha(v)beta(3) mediated process. These results indicate that hypoxia can increase expression of alpha(v)beta(3) in HUVECs, and that hypoxic regulation of alpha(v)beta(3) may be an important regulator of angiogenesis.     

Collagen synthesis by bovine aortic endothelial cells in culture.

Endothelial cells isolated from bovine aorta synthesize and secrete type III procollagen in culture. The procollagen, which represents the major collagenous protein in culture medium, was specifically precipitated by antibodies to bovine type III procollagen and was purified by diethyl-aminoethylcellulose chromatography. Unequivocal identification of the pepsin-treated collagen was made by direct comparison with type III collagen isolated by pepsin digestion of bovine skin, utilizing peptide cleavage patterns generated by vertebrate collagenase, CNBr, and mast cell protease. The type III collagen was hydroxylated to a high degree, having a hydroxyproline/proline ratio of 1.5:1.0. Pulse-chase studies indicated that the procollagen was not processed to procollagen intermediates or to collagen. Pepsin treatment of cell layers, followed by salt fractionation at acidic and neutral pH, produced several components which were sensitive to bacterial collagenase and which comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with alpha A, alpha B, and type IV collagen chains purified from human placenta by similar techniques. Bovine aortic endothelial cells also secreted fibronectin and a bacterial collagenase-insensitive glycoprotein which, after reduction, had a molecular weight of 135,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (using procollagen molecular weight standards) and which was not precipitable by antibodies to cold-insoluble globulin or to alpha 2-macroglobulin. Collagen biosynthesis by these cells provides an interesting model system for studying the polarity of protein secretion and the attachment of cells to an extracellular matrix. The presence of type III collagen in the subendothelium and the specific interaction of this protein with fibronectin and platelets suggest the involvement of this collagen in thrombus formation following endothelial cell injury.     

Posttranslational modifications in the biosynthesis of type IV collagen by a human tumor cell line

Factors responsible for the high extent of intracellular posttranslational modifications in type IV collagens were studied in a cultured human tumor cell line, HT-1080. These cells do not synthesize any detectable amounts of interstitial collagens but produce type IV collagen at a high rate, corresponding to about one-third of the production of interstitial collagens by cultured human skin fibroblasts. Prolyl 4-hydroxylase activity was lower in the HT-1080 cells than in human skin fibroblasts, there being a rough correlation between this enzyme activity and the rate of 4-hydroxyproline formation in these two cell types. The differing extents of the respective modifications could largely be explained by differences in the activities of lysyl hydroxylase and the hydroxylysyl glycosyltransferases between the two cell types. No difference ws found in prolyl 3-hydroxylase activity, however, even though the extent of 3-hydroxylation of proline residues was about 6-fold in the type IV collagens. In experiments where the HT-1080 cells were studied in suspension, a lag of about 100 min was found before the secretion of type IV collagen from the cells became linear. Pulse-chase experiments in suspension indicated that all the intracellular enzyme reactions proceeded for about 40 min, presumably due to the slow triple-helix formation in type IV collagens. This slow helix formation apparently contributed to the high extent of all the intracellular modifications but was not a major factor.     

Effect of vasopressin on type IV collagen production in human mesangial cells

Production of extracellular matrix proteins, such as type IV collagen and fibronectin, by mesangial cells contributes to progressive glomerulosclerosis. In this study, the ability of vasopressin (AVP), which causes mesangial cell proliferation and hypertrophy, to stimulate type IV collagen production by cultured human mesangial cells was examined using an enzyme-linked immunosorbent assay. AVP induced a concentration-dependent increase in the production of type IV collagen and this effect was potently and concentration-dependently inhibited by AVP V_1A receptor antagonists, including YM218. AVP also induced a concentration-dependent increase in transforming growth factor (TGF)-β secretion by human mesangial cells and this effect was inhibited by V_1A receptor antagonists. Furthermore, TGF-β also induced an increase in the production of type IV collagen; the AVP-enhanced production of type IV collagen was inhibited by an anti-TGF-β antibody. These findings indicate that AVP stimulates synthesis of type IV collagen by cultured human mesangial cells through the induction of TGF-β synthesis mediated by V_1A receptors; consequently, AVP contributes to glomerular remodeling and extracellular matrix accumulation observed in glomerular diseases.     

Alteration of the kinetics of type I procollagen synthesis in human osteosarcoma cells by 1,25-dihydroxyvitamin D3

The kinetics of type I procollagen synthesis in a human osteosarcoma cell line, MG 63, were investigated after treatment with 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂ D₃), a hormonal inducer of phenotypic differentiation. Pulse label and chase experiments demonstrated greatly enhanced production and more rapid reduction of intracellular procollagen molecules in the 1,25-(OH)₂ D₃–treated cells as compared to the nontreated case. After a chase for 1 h, labeled procollagen was reduced by nine-tenths in 1,25-(OH)₂ D₃–treated cells, while half of the radioactivity still remained in nontreated cells. The expression rate of type I collagen, which was examined by pulse label experiment, was elevated in association with an increase in the mRNA coding for the type I collagen α1 chain by 1,25-(OH)₂ D₃ treatment. However, the amount of intracellular procollagen present after 4 h continuous labeling was almost the same, independent of the 1,25-(OH)₂ D₃ treatment. Thus, we conclude that strage of the molecule was not affected. The results therefore suggest an increase in both the synthesis and secretion of type I collagen. The 1,25-(OH)₂ D₃ treatment was also found to induce the α subunit of prolyl 4-hydroxylase and to be associated with an elevated level of hydroxyproline in the procollagen. Moreover, gelatinase B–resistant procollagen molecules, indicative of intracellular procollagen molecules in the stable triple helical form, were detected only in the 1,25-(OH)₂ D₃–treated cells. These data suggest more efficient proline hydroxylation is involved in rapid secretion of procollagen after hormone administration. The present evidence points to posttranslational control of procollagen synthesis. J. Cell. Biochem. 65:542–549. © 1997 Wiley-Liss Inc.     

Fibrillar collagen type I stimulation of apolipoprotein B secretion in Caco-2 cells is mediated by β1 integrin

Caco-2 cells spontaneously differentiate into enterocyte-like cells and secrete apolipoprotein B (apoB) lipoproteins. We evaluated the effect of different extracellular matrix proteins on lipoprotein secretion by these cells. Caco-2 cells grown on human amnion connective tissue (HACT) secreted twice as much apoB as control cells on Transwells, but secreted similar amounts of apoA1. Cells cultured on fibrillar collagen type I secreted increased amounts of apoB similar to the cells cultured on HACT, but cells cultured on non-fibrillar collagen type I, type IV collagen or laminin-1 did not. The increased secretion was nullified by a function inhibiting anti-integrin β1 monoclonal antibody. Therefore, interactions between type I collagen and β1 integrins augment apoB secretion by Caco-2 cells. Cells on HACT formed a more uniform columnar epithelium with lipid droplets polarized to the basolateral membrane. We also studied the effect of extracellular matrix proteins on transepithelial resistance (TER) of differentiated Caco-2 cells. TER in cells cultured on HACT was similar to that on Transwells, but cells on laminin-1 and collagen IV exhibited higher TER. Thus, various extracellular matrix proteins regulate apoB secretion and TER differently. This new observation that extracellular matrix proteins can enhance apoB secretion in Caco-2 cells could be useful to explore the modulation of lipid transport by these proteins.