Modulation of proteoglycan metabolism by human fibroblasts maintained in an endogenous three-dimensional matrix.

This report describes synthesis and degradation of proteoglycans by human gingival fibroblasts growing in an endogenous three-dimensional matrix. Cells grown in the matrix cultures demonstrated a high rate of proteoglycan synthesis, varying between 2 and 4 times that of cells maintained in monolayer cultures. In addition, the relative amount deposited into the cell layer was increased in the matrix cultures, constituting 70% to 90% of the synthesized material during the first 24 h. Comparable levels for the monolayer cultures were 30% to 60%. The majority of the 35S-sulfate-labeled material in both matrix (80%) and monolayer (62%) cultures was susceptible to chondroitin ABC-lyase digestion. The major product was a low Mr (120,000) proteoglycan which could be immunoprecipitated by an antibody against PGII (decorin). In addition, the cells synthesized two chondroitin ABC-lyase-sensitive proteoglycans, one with Mr greater than 400,000, one with an apparent Mr of 250,000, as well as two heparan sulfate proteoglycans with Mr greater than 250,000. The low Mr dermatan sulfate, decorin, was also the major component deposited in the three-dimensional matrix, constituting about 60% of the total sulfate incorporation. In contrast, fibroblasts in monolayer cultures deposited only a small amount (13%) of decorin (PGII) in the cell layer, and the major proteoglycan in this compartment was heparin sulfate. The rate of release of the newly deposited proteoglycans was the same in the two culture conditions, although material released from the three-dimensional matrix cultures contained small Mr components indicating a higher degree of degradation. These studies show differences in proteoglycan metabolism by gingival fibroblasts grown in an endogenous matrix and in monolayer cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dermatan sulphate proteoglycans from sclera examined by rotary shadowing and electron microscopy.

Two dermatan sulphate-containing proteoglycans from bovine sclera were examined by rotary shadowing and electron microscopy, and the results were compared with previous biochemical findings. Both the large iduronate-poor proteoglycan (PGI) and the small iduronate-rich proteoglycan (PGII) possessed a globular proteinaceous region. Whereas PGI had a branched extension from the globular region, with five to eight side chains attached to it, PGII had only a single tail, which was of glycosaminoglycuronan. PGII aggregated via globular-region interactions, which were much diminished by reduction and alkylation. PGI aggregated via side chains and globular-region interactions. Although a few PGI aggregates were large, and similar to the hyaluronan-cartilage proteoglycan aggregates [Weidemann, Paulsson, Timpl, Engel & Heinegård (1984) Biochem. J. 224, 331-333], hyaluronan did not cause enhanced aggregation. PGII is very similar in shape to the small cartilage chondroitin sulphate proteoglycan, whereas PGI somewhat resembles the large cartilage chondroitin sulphate proteoglycan, although with many fewer glycosaminoglycan side chains, and probably only one globular region as opposed to two in the cartilage proteoglycan.     

Modulation of types I and III procollagen synthesis at various stages of arterial smooth muscle cell growth in vitro

Collagen synthesis was monitored in cultures of rabbit arterial smooth muscle cells (SMC). Both the rate of collagen synthesis per cell and collagen synthesis as a percent of total protein synthesis were measured at specific intervals from 1 to 14 days after inoculation of smooth muscle cells. The proportions of types I and III collagen present in the conditioned incubation medium and in the cell layer were also examined. After inoculation the cells displayed population expansion typical of SMC in which growth slowed but did not cease after the cells attained confluence. Collagen synthesis rates, expressed as [14C]hydroxyproline per cell, were eight-fold higher in preconfluent cells. In these cultures collagen accounted for more than 20% of the newly synthesized, 14C-labeled protein present as trichloroacetic acid (TCA)-insoluble material in 24 h culture media. In post-confluent cultures, this percentage was reduced to about 7% of the total protein synthesized. Synthesis rates of both collagen and non-collagen protein decreased with increasing time after inoculation. However, the rate of decline of collagen synthesis was three times greater than that seen for non-collagen protein. Early cultures synthesized relatively more type I than type III procollagen. The type I to type III ratio was highest at day 3 and declined after that time to day 14. While the synthesis of both types decreased with increasing age, type I declined at a greater rate resulting in a predominance of type III procollagen secretion by older cultures. We conclude that protein synthesis in general and collagen synthesis in particular are quantitatively and qualitatively dependent upon the growth stage of SMC in vitro.     

Interstitial collagen synthesis by somatic testicular cells in culture

The synthesis, distribution and types of collagen produced by somatic testicular cells in culture was studied. To investigate whether changes in collagen synthesis correlate with the age of the animal, cultures derived from immature and pubertal rats were established. Immature rats synthesize 40 per cent more collagen than pubertal rats. Both groups of animals synthesize procollagen types I and III. Pro-collagen type I is present in the culture medium as well as in the cell fraction, while type III is only detected in the culture medium. In the transition from immature to pubertal rat, the ratio of procollagen type III to procollagen type I diminishes from 5.7 to 1.7. These results indicate that the synthesis, distribution and molecular characteristics of interstitial collagens changes with the age of the animal. Since, the content of other extracellular matrix components such as proteoglycans and collagen type IV also varies with age, we postulate that the composition of the extracellular matrix in the testes is not constant but changes with sexual development.     

Proteoglycans from pig aorta. Comparative study of their interactions with lipoproteins.

Different proteoglycans (PGs) were isolated from pig aorta for aggregation studies with hyaluronic acid and human low-density lipoproteins (LDL). Extraction of the intima-media with 4M-guanidinium chloride and digestion of the residue with collagenase solubilized 91% of aortic hexuronic acid content. From the guanidinium chloride extract two PGs were isolated by ion-exchange and gel-permeation chromatography: proteochondroitin sulphate (PGI) with a protein-core apparent Mr of 250 000 and proteodermatan-chondroitin sulphate (PGII) with a protein-core apparent Mr of 55 000. Only PGI forms high-Mr aggregates with hyaluronic acid. From the collagenase digest two other PGs were isolated: proteoheparan sulphate and proteochondroitin sulphate (PGIII and PGIV respectively). PGIV had a smaller hydrodynamic size than PGI. PGI and PGII formed insoluble complexes with human LDL in the presence of Ca2+. PGIII or PGIV did not form precipitates with the LDL. PGI and PGII, but neither PGIII nor PGIV, were bound to LDL-Sepharose. The main peaks of PGI and PGII were eluted from LDL-Sepharose with 60 mM- and 90 mM-NaCl respectively. The results indicate that aortic PGs have different interacting potentials with lipoproteins, depending on their Mr and their glycosaminoglycan composition.     

Pulmonary macrophages alter the collagen phenotype of lung fibroblasts

Cultured lung fibroblasts produced and secreted interstitial collagen types I and III. The relative proportion of type III collagen increased as a linear function of cell density, with confluent cultures producing 8.6% type III collagen. When human lung fibroblasts were cultured in the presence of newly harvested lung macrophages, the proportion of type III collagen secreted rose to 15.5%. This high level of type III collagen synthesis was greater than could be induced by withdrawal of serum, a perturbation known to alter the proportion of types I and III collagen synthesized by fibroblasts. This effect on fibroblast phenotype was independent of cell density, as both low and high density cultures of fibroblasts responded similarly when cultured with macrophages. There was no evidence that fibroblasts synthesize new or different collagen types (such as type I trimer) in response to macrophages. Optimal conditions for eliciting an effect on fibroblast connective tissue metabolism required interaction of the two cell types for 5–8 days. These in vitro changes are analogous to the sequence of interactions and changes in connective tissue metabolism seen during recovery from tissue injury.     

Proteoglycan synthesis by fibroblast cultures initiated from regions of adult bovine tendon subjected to different mechanical forces

Fibroblast cultures were initiated from two distinct regions of the adult bovine deep flexor tendon and synthesis of 35S-labeled proteoglycans by these cultures was investigated. The proximal/tensional region of the tendon was composed of linearly arranged dense collagen bundles, and its glycosaminoglycan hexosamine content was only 0.2% of the dry weight of the tissue. The proteoglycans of this region were predominantly small (Kav = 0.5 on Sepharose CL-4B). Cells placed into culture from this region attached to the substratum readily, and the radiolabeled proteoglycans from these cultures were 90% small proteoglycans. In a more distal region of the tendon that is subjected to compressive forces, the collagen was arranged as a network of fibrils separated from each other by a matrix that stained intensely with Alcian blue. The glycosaminoglycan content of this compressed region was up to 5-fold higher than in the proximal region, and as much as 50% of the proteoglycans were large molecules (eluted from Sepharose CL-4B in the Vo). Cells placed into culture from the distal/compressed region did not attach to the substratum as readily as those from the proximal region and were characterized by the presence of numerous cytoplasmic lipid inclusions. The [35S]proteoglycans synthesized by the distal tendon fibroblast cultures were divided into two approximately equal populations of large and small proteoglycans having elution characteristics similar to the proteoglycans extracted from this tissue. The distinct profiles of proteoglycan production were maintained by the cells in culture for several weeks, although eventually the amount of large proteoglycan synthesized by the distal tendon fibroblast cultures diminished. Both regions of tendon contained predominantly type I collagen, and collagen production was about 10% of the total protein synthesized by both cell cultures. These observations indicate that adult tendon fibroblasts in culture express stable synthesis of proteoglycan populations similar to those found in the region of tendon from which they were derived.     

Turnover of proteoglycans by chick lens epithelial cells in cell culture and intact lens

Chick lens epithelial cells were cultured on plastic and type IV collagen substrata, and the confluent cultures were labeled continuously with [35S]sulfate for 20 h. Intact lenses were also labeled in the same way. 35S-Proteoglycans isolated from those cultures were compared for their molecular sizes and glycosaminoglycan compositions. The results have shown that: 1) Proteoglycans synthesized by cells on type IV collagen were significantly smaller than those by cells on plastic. 2) Proteoglycans of intact lens showed a broad distribution of molecular size and contained a high proportion of chondroitin sulfate in the medium fraction compared to those of the two cell cultures. In order to explain such differences between proteoglycans from cultures, label-chase experiments with [35S]sulfate were done for proteoglycans synthesized. 35S-Proteoglycans isolated at each chase time 0, 2.5, and 17 h) were compared and the following results were found: 1) The cell layers of both "plastic" and "type IV collagen" cultures contained glycosaminoglycan species predominantly at each chase time rather than proteoglycans. 2) Changes in the glycosaminoglycan compositions of medium fractions of cell cultures were observed during the chase period; in medium of the "plastic" culture, proteoheparan sulfate increased with chase time, whereas in medium of the "type IV collagen" culture, chondroitin sulfate glycosaminoglycan (not proteoglycan) increased with chase time. 3) In intact lens culture, lens capsule fraction at every chase time contained a proteoglycan unique in molecular size, which was not found in cell culture fractions. 4) All fractions from intact lens cultures contained a higher content of chondroitin sulfate at every chase time than the respective fractions from cell cultures. These results suggest that adhesion of the cells to type IV collagen or lens capsule influences the degradation and secretion of proteoglycans. In addition, they can account partially for the above-described differences in molecular sizes and glycosaminoglycan compositions between 35S-proteoglycans from various cultures continuously labeled with [35S]sulfate.     

Influence of cell density on collagen biosynthesis in fibroblast cultures.

Characteristic features of collagen metabolism in human skin fibroblasts were studied in relation to cell density. Measuring peptide-bound hydroxyproline we found that collagen synthesis per cell decreased when cultures approached confluency. On the other hand, the relative rate of collagen synthesis (collagen/total protein) was higher in quiescent than in proliferating cultures. With increasing cell density the proportion of type III collagen in comparison with type I was found to be slightly increased. In addition, in low-density cultures [alpha I(I)]3 collagen trimers were produced in considerable amounts, whereas they were no longer detected in cultures with a high cell density. Although hydroxylation of proline residues was normal in all cell stages, conversion of procollagen into collagen was found to depend strongly on the density at which the cells were investigated. Almost no cleavage of procollagen peptides was observed in rapidly growing cells, whereas highly confluent cell cultures converted most of the newly synthesized procollagen molecules.     

Influence of aging on glycosaminoglycans and small leucine-rich proteoglycans production by skin fibroblasts

Skin aging is characterised by a progressive deterioration of its functional properties, linked to alterations of dermal connective tissue. Whereas many studies have been devoted to collagen alterations during aging, the situation is less clear concerning glycosaminoglycans and proteoglycans. Particularly, the alterations of the expression of small leucine-rich proteoglycans (SLRPs), a family of proteoglycans strongly implicated in cell regulation, have never been studied.In the present study we measured glycosaminoglycans and small leucine-rich proteoglycans synthesis by skin fibroblasts from donors of 1 month to 83 years old. [³H]-glucosamine and [³⁵S]-sulfate incorporation did not show significant differences of sulfated GAG synthesis during aging. On the other hand, a significant positive correlation was found between hyaluronan secretion and donor’s age. Northern blot analysis of SLRPs mRNAs showed a significant negative correlation of lumican mRNA with donor’s age, whereas decorin and biglycan mRNAs were not significantly altered. Immunohistochemical study and quantitative image analysis confirmed a decreased lumican accumulation in aged human skin.Taken together, our results suggest that impairment of glycosaminoglycans and SLRPs synthesis might be involved in the functional alterations of aged skin.     

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.     

Ovarian carcinoma cells synthesize both chondroitin sulfate and heparan sulfate cell surface proteoglycans that mediate cell adhesion to interstitial matrix

Metastatic ovarian carcinoma metastasizes by intra-peritoneal, non-hematogenous dissemination. The adhesion of the ovarian carcinoma cells to extracellular matrix components, such as types I and III collagen and cellular fibronectin, is essential for intra-peritoneal dissemination. The purpose of this study was to determine whether cell surface proteoglycans (a class of matrix receptors) are produced by ovarian carcinoma cells, and whether these proteoglycans have a role in the adhesion of ovarian carcinoma cells to types I and III collagen and fibronectin. Proteoglycans were metabolically labeled for biochemical studies. Both phosphatidylinositol-anchored and integral membrane-type cell surface proteoglycans were found to be present on the SK-OV-3 and NIH:OVCAR-3 cell lines. Three proteoglycan populations of differing hydrodynamic size were detected in both SK-OV-3 and NIH:OVCAR-3 cells. Digestions with heparitinase and chondroitinase ABC showed that cell surface proteoglycans of SK-OV-3 cells had higher proportion of chondroitin sulfate proteoglycans (75:25 of chondroitin sulfate:heparan sulfate ratio), while NIH:OVCAR-3 cells had higher proportion of heparan sulfate proteoglycans (10:90 of chondroitin sulfate:heparan sulfate ratio). RT-PCR indicated the synthesis of a unique assortment of syndecans, glypicans, and CD44 by the two cell lines. In adhesion assays performed on matrix-coated titer plates both cell lines adhered to types I and III collagen and cellular fibronectin, and cell adhesion was inhibited by preincubation of the matrix with heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, or chondroitin glycosaminoglycans. Treatment of the cells with heparitinase, chondroitinase ABC, or methylumbelliferyl xyloside also interfered with adhesion confirming the role of both heparan sulfate and chondroitin sulfate cell surface proteoglycans as matrix receptors on ovarian carcinoma cells.     

Ascorbate-independent proline hydroxylation resulting from viral transformation of Balb 3T3 cells and unaffected by dibutyryl cAMP treatment.

Collagen synthesis, hydroxylation of proline in collagen, and collagen secretion were studied in the contact-inhibited mouse fibroblast line, Balb 3T3; the Kirsten virus transformed line, Ki-3T3; and dibutyryl cAMP (dbcAMP)-treated Ki-3T3 cells, during the various phases of the growth cycle. Transformed cells in both logarithmic and stationary phase produced lower levels of collagen than the parent line but 85-90% of the theoretically possible hydroxyproline residues of the collagen were formed even when ascorbic acid was not added to the culture medium. Moreover, the transformed cells showed only about a 20% increase of collagen secretion upon addition of ascorbate. This was in contrast to the ascorbate requirement for maximal proline hydroxylation and the 2-3 fold stimulation of collagen secretion by ascorbate in the parent Balb 3T3 cells. Although dbcAMP treatment caused Ki-3T3 cells to assume a more normal morphology and increased the relative rate of collagen synthesis to levels similar to that of 3T3, such treatment did not restore an ascorbate requirement for proline hydroxylation or collagen secretion. The specific activity of the enzyme prolyl hydroxylase also was not affected by dbcAMP treatment although collagen synthesis was increased by such treatment. In addition, it was found that ascorbic acid was not effective in activating prolyl hydroxylase derived from Ki-3T3 or dbcAMP-treated Ki-3T3 cell cultures either in logarithmic phase or stationary phase. Ki-3T3 cultures did not accumulate ascorbic acid in cells or medium nor was ascorbic acid synthesized from the precursor 14C-glucuronate in cell homogenates. The results suggest that virally transformed Balb 3T3 cells acquire the capacity to synthesize a reducing cofactor for prolyl hydroxylase and that this function may be related to the increased glycolytic metabolism of these cells since neither cellular metabolism nor ascrobate-independent hydroxylation was altered by treatment with dbcAMP.     

Comparison of the collagenous products synthesized in culture by pig aortic endothelial and smooth-muscle cells. Variability in endothelial-cell cultures.

In contrast with smooth-muscle cells from the same tissue, endothelial cells from pig aorta were found to exhibit in culture considerable variability in the pattern of collagen synthesis between one isolation of cells and the next. Synthesis varied from largely collagen type I to virtually all type III in the absence of type I but with small amounts still of collagens types IV and V, to, in one instance, synthesis basically of only type V. Synthesis usually by these cells of collagen predominantly of the interstitial type (I and III) rather than, as might be expected, that from basement membrane (type IV) was not attributable to the influence of subculture. All four collagen types were deposited in the cell layer to an increased extent in primary compared with secondary cultures of either smooth muscle or endothelial origin. Endothelial cells appeared sometimes to synthesize a large-Mr collagenous entity that might conceivably be related to 'short-chain' collagen. In addition, small-Mr hydroxyproline-containing peptides were detected that might reflect rapid collagen(s) turnover in endothelial cultures.     

Donor-age-related changes in the morphology, growth potential, and collagen biosynthesis in rat fibroblast subpopulations in vitro

Primary explant, mass and clonal fibroblast cultures established from BN rat skin and lungs were used to examine changes in cell behaviour associated with aging. Three distinct fibroblast cell types, i.e. FI, FII and FIII, could be identified on the basis of their morphological and proliferative properties. They could also be distinguished from each other by the amount and type of collagen they synthesized in clonal cultures. FI cells are diploid, spindle shaped and highly proliferative, and they synthesize low levels of type-I and -III collagen. Epithelioid FII cells are also diploid, proliferate slowly, and exhibited elevated collagen synthesis as compared to FI cells. FIII fibroblasts are large, stellate, tetraploid cells that proliferate more slowly than the other types but synthesize large amounts of collagen. In comparison to FI cells, the level of type-III-collagen synthesis is slightly elevated in FIII fibroblasts. In primary explant, mass and clonal cultures, the relative proportions of FI, FII and FIII cells were found to change as a function of the age of the donor animal. The increasing predominance of FIII cells in mass cultures of fibroblasts obtained from donors of increasing ages was consistent with the increased level of collagen synthesis in these cultures. Our observations indicate that the differentiation of normal BN rat fibroblasts occurs via a three-phase process. We discuss the age-related changes in the relative abundance of FI, FII and FIII cells in vivo in the context of alterations in fibroblast replacement and changes in connective tissue that occur during aging.     

Differential regulation of extracellular matrix proteoglycan (PG) gene expression. Transforming growth factor-beta 1 up-regulates biglycan (PGI), and versican (large fibroblast PG) but down-regulates decorin (PGII) mRNA levels in human fibroblasts in culture

Proteoglycans (PGs) comprise a group of extracellular matrix macromolecules which play an important role in matrix biology. In this study, normal human skin and gingival fibroblast cultures were incubated with transforming growth factor-beta 1 (TGF-beta 1), and the expression of three PGs, viz. biglycan (PGI), decorin (PGII), and versican (a large fibroblast proteoglycan) was examined. The results indicate that TGF-beta 1 (5 ng/ml) markedly increased the expression of biglycan (up to 24-fold) and versican (up to 6-fold) mRNAs and the enhancement of biglycan expression was coordinate with elevated type I procollagen gene expression in the same cultures. In contrast, the expression of decorin mRNA was markedly (up to approximately 70%) inhibited by TGF-beta 1. The response to TGF-beta 1 was similar in both skin and gingival fibroblasts, although the gingival cells were clearly more responsive to stimulation by TGF-beta 1 with respect to biglycan gene expression. Analysis of 35S-labeled proteoglycans in the culture media of skin and gingival fibroblasts also revealed stimulation of biglycan and versican production, and reduction in decorin production. Quantitation of both [35S]sulfate and [3H]leucine-labeled decorin in cell culture media by immunoprecipitation revealed a 50% reduction in decorin production in cell cultures treated with TGF-beta 1. This TGF-beta 1-elicited reduction was accompanied by an apparent increase in the size of the decorin molecules, although the size of the core protein was not altered, as judged by Western immunoblotting following chondroitinase ABC digestion. Analysis of the proteoglycans in the matrix and membrane fractions also revealed increased amounts of versican in cultures treated with TGF-beta 1. These results indicate differential regulation of PG gene expression in fibroblasts by TGF-beta 1, and these observations emphasize the role of PGs in the extracellular matrix biology and pathology.     

Localization of PGI (biglycan, BGN) and PGII (decorin, DCN, PG-40) genes on human chromosomes Xq13-qter and 12q, respectively

The genes for PGI (biglycan, BGN) and PGII (decorin, DCN) have been assigned to human chromosomes X and 12 by Southern analysis of a panel of human-rodent somatic cell hybrid DNAs with cDNA probes for these related small proteoglycans. Regional localization of BGN to Xq13-qter and DCN to 12p12.1-qter was also obtained by examining hybrids containing spontaneous breaks or well-characterized translocations involving chromosomes X and 12. Biglycan (BGN) is a single-copy gene about 6 kb in length. Hybridization with subfragment cDNA probes suggests the presence of two copies of the decorin (DCN) gene, or related sequences, at the locus on chromosome 12, although there is no evidence for function of more than one DCN gene. Efforts to detect restriction fragment length polymorphisms with these probes were unsuccessful.     

Evidence for independent metabolism and cell surface localization of cell surface localization of cellular proteoglycans and glycosaminoglycan free chains

The synthesis and turnover of metabolically labeled proteoglycans from medium, cell layer, and substratum-associated compartments were characterized in four cell lines of fibroblastic origin, including a fibrosarcoma line, and in the murine melanoma cell type, B16.F10. Substantial differences were apparent between the various cell types with regard to quantities, hydrodynamic sizes, and compartmentalization of labeled product. Such variations were greater between the different cell lines than between separately labeled cultures of the same cell type. Greater than 85% of cell-associated proteoglycans were accessible to glycosaminoglycan-degrading enzymes added to the medium of monolayer cultures, demonstrating their principal location to be external to the cell membrane. Apparent glycosaminoglycan free chains, determined by a lack of change in hydrodynamic size following alkaline elimination, were among the products from each cell line and were similarly found to be in a principally pericellular location. Results from label-chase studies demonstrated apparent independent kinetics for proteoglycans and glycosaminoglycan free chains, with little conclusive evidence for precursor-product relationships. Also, their processing by the cells was different, since the proteoglycans were shed largely unchanged into the medium for the three cell lines evaluated, whereas the free chains were not recoverable from the medium in significant amounts. The latter observation suggests the internalization of cell surface-associated free chains and their depolymerization at an intracellular site. The results, which indicate that the content, cellular disposition, and turnover of proteoglycans are quite variable between the cell lines studied, may reflect fundamental cell type-specific specialization in the metabolism of these complex substances. Furthermore, the data raise the interesting possibility that glycosaminoglycan free chains may have biological functions at the cellular level, independent of proteoglycans.     

Synthesis of types I and III procollagen and collagen by monkey aortic smooth muscle cells in vitro.

Analysis of pepsin-resistant proteins produced in culture by monkey aortic smooth muscle cells (SMC) indicates the synthesis of types I and III collagen. As determined by carboxymethylcellulose chromatography and disc gel electrophoresis, SMC cultures synthesize more type III collagen than monkey skin fibroblast cultures; aortic adventitial cell cultures (a mixture of SMC and fibroblasts) synthesize an intermediate amount of type III collagen. Both types I and III procollagens can also be isolated from the culture medium of SMC and skin fibroblasts. The procollagens were separated by diethylaminoethylcellulose (DEAE-cellulose) chromatography in identified by electrophoresis and after cleavage with pepsin and cyanogen bromide. Quantitation of the procollagen by DEAE-cellulose chromatography suggests that 68% of the SMC procollagens and less than 10% of the skin fibroblast procollagens are type III. On the other hand, estimation of the proportions of collagen types secreted by cells, employing pepsin digestion of cell culture medium at 15 degrees C, leads to an underestimation of the amount of type III collagen relative to type I. SMC and fibroblasts may differ in their ability to convert type I procollagen to collagen ad indicated by the observation that skin fibroblast culture medium contains both pN and pC collagen intermediates after 24 h, while cultures of SMC essentially lack the pC collagen intermediates.     

Biochemical comparison of fibroblast populations from different periodontal tissues: characterization of matrix protein and collagenolytic enzyme synthesis

To compare the expression of extracellular matrix components by fibroblasts from different periodontal tissues, rat molar periodontal ligament fibroblasts (RPL) and rat gingival fibroblasts (RGF) were isolated and cultured from individual animals. Pulse-chase experiments using [35S]methionine as a precursor revealed that confluent populations of early passage cells of both cell types synthesized similar amounts of collagen, fibronectin, and SPARC/osteonectin. Qualitative and quantitative differences were apparent in the relative proportions of type III collagen, in the rates of procollagen processing, and in the synthesis of a small number of unidentified proteins observed by sodium dodecyl sulphate--polyacrylamide gel electrophoresis. Collagen constituted 24-26% of the radiolabelled proteins secreted by both cell types, type I being the predominant collagen, with lower amounts of type III (3-8% RGF, 8-18% RPL) and type V (approximately 1%) collagens. Procollagen processing in the culture medium of RPL cells was more rapid than for RGF cells, but was increased in multilayered cultures of both RPL and RGF. In multilayered cultures, collagen TCA fragments, indicative of tissue collagenase activity, were also identified. Active and latent tissue collagenases and a latent form of a novel collagenolytic enzyme (matrix metalloendoproteinase-V) that cleaves native TCA fragments were demonstrated in these cultures. Addition of either concanavalin A (10(-6) M) or retinoic acid (10(-5) M) to the culture medium stimulated the secretion of the latent collagenolytic enzymes. Collagenase inhibitor was also synthesized by both RGF and RPL cells. SPARC/osteonectin, a 40-kilodalton glycoprotein, represented 0.5-1.0% of the secreted radiolabelled proteins of both cell types.