Characterization of proteoglycans synthesized by fetal rat lung type II pneumonocytes in vitro and the effects of cortisol

The synthesis of proteoglycans by primary cultures of 19-day gestation fetal rat lung Type II pneumonocytes was studied. The cells were grown in the presence of [3H]-glucosamine and/or [35S]-Na2SO4 and the radioactive label incorporated into proteoglycans was analyzed. Proteoglycans of high molecular weight (approximately 200 Kd) were isolated by gel permeation chromatography and contained both [3H] and [35S]. The glycosaminoglycan composition of the proteoglycans was determined by electrophoresis and autoradiography. The medium contained 65-80% of the labeled proteoglycans and was enriched for hyaluronate, with lesser amounts of the sulfated glycosaminoglycans (dermatan sulfate greater than heparan sulfate greater than chondroitin sulfate). The cell layers retained 20-35% of the labeled proteoglycans and was enriched for heparan sulfate, with lesser amounts of chondroitan sulfate greater than dermatan sulfate greater than hyaluronate. The synthesis of proteoglycans was time-dependent and was stimulated by increasing concentrations of fetal bovine serum. Cortisol inhibited proteoglycan synthesis, apparently by decreasing the availability of proteoglycan core-protein.     

The synthesis of proteoglycans in fat-storing cells of rat liver

Fat-storing cells (perisinusoidal stellate cells) were isolated by enzymatic digestion of rat liver and purified by a single-step Nycodenz gradient to yield 11.4 X 10(6) cells per liver, with a purity of 74% and a viability of 76%. Monolayer cultures of fat-storing cells incorporated both [35S]sulfate and [3H]glucosamine into glycosaminoglycans; the rate of incorporation increased with culture time (3-fold between the third and eighth days in culture). About 80% of newly formed glycosaminoglycans were secreted into the medium. Analysis of the types of glycosaminoglycans revealed a different pattern for cells and medium, respectively, which is subject to culture time. Heparan sulfate remains primarily cell-bound and, therefore, has a low fractional secretion rate. Chondroitin sulfate and even more dermatan sulfate are the main types of glycosaminoglycans in the medium. Dermatan sulfate represents about 60% of total medium glycosaminoglycans. In advanced cultures (eighth day), this type becomes the predominant one in the cell layer. The reduction of the molecular weight of native medium-sulfated molecules by papain digestion and beta-elimination and the puromycin-induced inhibition of their synthesis by more than 75% suggest the formation of glycosaminoglycans as complex proteoglycans. It is concluded that fat-storing cells are a major cellular source of dermatan sulfate and chondroitin sulfate in liver connective tissue. Since the pattern of proteoglycans of fat-storing cells closely resembles that found in the fibrotic liver matrix, this cell type might be of pathogenetic significance for the accumulation of chondroitin sulfate and dermatan sulfate in cirrhotic connective tissue.     

Kupffer cell-mediated induction of synthesis and secretion of proteoglycans by rat liver fat-storing cells in culture

The effect of conditioned media from Kupffer cells of normal, D-galactosamine- and thioacetamide-treated rats on the synthesis of proteoglycans by rat liver fat-storing cells in culture was studied in order to elucidate some of the mechanisms initiating enhanced connective tissue proteoglycan synthesis in injured liver. The incorporation of [35S]sulfate and [3H]glucosamine into proteoglycans was 2.1-2.5 fold (P less than 0.005) stimulated by the additions of normal, D-galactosamine- and thioacetamide-exposed Kupffer cell media. The concentrations of hexuronic acid and amino sugars in the medium glycosaminoglycan fraction were enhanced 5-fold and 4.5-fold, respectively, if the fat-storing cells were cultured in the presence of normal Kupffer cell conditioned medium. Treatment of normal Kupffer cells in culture with zymosan and phorbol esters, but not the addition of lipopolysaccharide, enhanced further the proteoglycan synthesis-stimulating effect of normal (untreated) Kupffer cells. The pattern of newly formed [35S]sulfate-labeled proteoglycans was changed in the presence of Kupffer cell media, showing a strong fractional increase of chondroitin sulfate and a relative decrease of dermatan sulfate, but the fraction of heparan sulfate was almost unaffected. In absolute terms Kupffer cells stimulated the total (medium and cell fraction) synthesis of chondroitin sulfate 2.8-fold and that of dermatan sulfate 1.5-fold. Although the DNA content of fat-storing cell cultures was increased by incubation with Kupffer cell media, an enhancement of proteoglycan synthesis was also observed when related to the DNA content of the cultures. The stimulation of proteoglycan synthesis was not dependent on the induction of cell proliferation. Gel chromatography and beta-elimination of medium proteoglycans revealed no changes of the molecular weight distribution profile of native proteoglycans and glycosaminoglycan chains synthesized under the influence of the various Kupffer cell media. Activation of proteoglycan synthesis and secretion in fat-storing cells by Kupffer cell-derived factor(s) might be an important mechanism of their strong accumulation in the connective tissue of fibrotic livers.     

Changes of glycosaminoglycan synthesis during in vitro ageing of human fibroblasts (WI-38).

Synthesis rates of glycosaminoglycans by WI-38 cultures (diploid, human fibroblasts exhibiting a limited number of population doublings in vitro) were determined by incorporation of 35S-sulfate of 14C-glucosamine into cellular and extracellular glycosaminoglycans at different passage levels before phase out. A progressive decline in the synthesis of cellular and extracellular glycosaminoglycans occured during the last (about 4) population doublings. 35S-sulfate incorporation into extracellular glycosaminoglycans appeared to be somewhat more reduced than 14-C-glucosamine incorporation during the last passages. Analysis of the distribution pattern of incorporated label into various glycosaminoglycan types (hyaluronic acid, chondroitin sulfate, dermatan sulfate and possibly heparan sulfate) revealed an age-related relatively stonger decline of 14C-glucosamine incorporation into cellular and extracellular hyaluronic acid and of 35S-sulfate into extracellular chondroitin sulfate in comparison with the other glycosaminoglycan types. Addition of exogenous glycosaminoglycans (chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, hyaluronic acid, heparan sulfate, heparin) at 100 microgram/ml to the culture media during the last 7 to 10 population doublings before phase out did not increase the total number of population-doublings. Heparin exhibited a significant growth inhibitory effect at 100 or 500 microgram/ml. The changes in glycosaminoglycan metabolism are interpreted as an expression of cellular ageing, and such an in vitro system offers a model for analyzing the factors involved in or causing the induction respectively prevention of this functional change.     

Effects of limited exposure of rabbit chondrocyte cultures to parathyroid hormone and dibutyryl adenosine 3',5'-monophosphate on cartilage-characteristic proteoglycan synthesis

Treatment of rabbit chondrocyte cultures with PTH or (Bu)2cAMP for 30 h increased by 2- to 3-fold the incorporation of [35S]sulfate and 3H radioactivity with glucosamine as the precursor into large chondroitin sulfate proteoglycans characteristically found in cartilage matrix. However, PTH and (Bu)2cAMP did not increase either [35S]sulfate incorporation into small proteoglycans or the incorporation of 3H radioactivity into hyaluronic acid and other glycosaminoglycans. PTH and (Bu)2cAMP also increased the incorporation of [3H] serine into both proteoglycans and total protein. In all cultures described above, the stimulation of [3H]serine incorporation into proteoglycans exceeded that of [3H]serine incorporation into total protein. These data indicate that PTH and (Bu)2cAMP selectively stimulate cartilage proteoglycan synthesis while they increase total protein synthesis. Since cAMP seems to play a mediatory role in the action of PTH, we elected to examine the effects of a limited exposure of chondrocytes to PTH or (Bu)2cAMP on the synthesis of proteoglycans. Treatment with PTH or (Bu)2cAMP for only the initial 2-7 h did not increase the rates of incorporation of [35S]sulfate, the 3H radioactivity with glucosamine, and [3H]serine into proteoglycans, as measured at 30 h, despite the fact that this treatment brought about a rapid and transient rise in the cAMP level. Furthermore, the application of prostaglandin I2 at concentrations that increased cAMP levels in a similar fashion as did PTH did not affect [35S] sulfate incorporation into proteoglycans. These observations suggest that in addition to the transient rise of cAMP, other biochemical changes are required for elaboration of the effect of PTH on proteoglycan synthesis. Although cAMP analogs mimic some of the effects of PTH in chondrocytes, the nucleotides and PTH appear to stimulate proteoglycan synthesis by different mechanisms.     

Identification of chondroitin sulfate E proteoglycans and heparin proteoglycans in the secretory granules of human lung mast cells

The predominant subclasses of mast cells in both the rat and the mouse can be distinguished from one another by their preferential synthesis of 35S-labeled proteoglycans that contain either heparin or oversulfated chondroitin sulfate glycosaminoglycans. Although [35S]heparin proteoglycans have been isolated from human lung mast cells of 40-70% purity and from a skin biopsy specimen of a patient with urticaria pigmentosa, no highly sulfated chondroitin sulfate proteoglycan has been isolated from any enriched or highly purified population of human mast cells. We here demonstrate that human lung mast cells of 96% purity incorporate [35S] sulfate into separate heparin and chondroitin sulfate proteoglycans in an approximately equal to 2:1 ratio. As assessed by HPLC of the chondroitinase ABC digests, the chondroitin [35S]sulfate proteoglycans isolated from these human lung mast cells contain the same unusual chondroitin sulfate E disaccharide that is present in proteoglycans produced by interleukin 3-dependent mucosal-like mouse mast cells. Both the chondroitin [35S]sulfate E proteoglycans and the [35S]heparin proteoglycans were exocytosed from the [35S]sulfate-labeled cells via perturbation of the IgE receptor, indicating that both types of 35S-labeled proteoglycans reside in the secretory granules of these human lung mast cells.     

Influence of collagen substrata on glycosaminoglycan production by B16 melanoma cells.

A cloned metastatic murine melanoma cell line exhibited similar growth characteristics when propagated on either type I collagen, type IV collagen, or plastic. However, cells grown on both types of collagen exhibited an altered cellular morphology and on type IV collagen only, an increased substrate adhesiveness, relative to those maintained on a plastic substratum. Incorporation of [3H]glucosamine and [35S]sulfate into glycosaminoglycans (GAGs) of cells grown on collagen substrates was 20% and 40% less, respectively, than cells grown on plastic, whereas degradation of cell-associated [35S]sulfate-labeled GAGs was similar in cells grown on collagen or plastic. Although the composition of GAGs was similar in all cultures, consisting of approximately 60% chondroitin and 40% heparin or heparan sulfate, the degree of sulfation of the heparin or heparan sulfate molecules was markedly decreased in cultures grown on collagen. The results indicate that the composition of the extracellular matrix influences the biological behavior of B16 melanoma cells, in part by altering the amount and nature of the GAG molecules produced.     

Independent and synergistic actions of somatomedin-C in the stimulation of proteoglycan biosynthesis by cultured rat granulosa cells

The role of somatomedin-C (Sm-C) in the regulation of granulosa cell proteoglycan biosynthesis was investigated in vitro in a primary culture of rat granulosa cells labeled with [35S]sulfate. Basal [35S]sulfate incorporation into extracellular proteoglycans was increased by 93 percent in response to treatment with highly purified Sm-C (50 ng/ml) by itself. Whereas treatment with a minimally effective dose of FSH (20 ng/ml) alone produced a 43 percent increase over basal levels in extracellular [35S]sulfate-labeled proteoglycans, concurrent treatment with Sm-C yielded a 2.7-fold amplification of the FSH effect. Qualitatively similar results were obtained when [35S]sulfate incorporation into cellular proteoglycans was determined, the latter accounting for approximately one half of the total radioactivity incorporated. Significantly, fractionation of the major extracellular proteoglycan species revealed FSH to favor the exclusive production of dermatan sulfate (1.6-fold increase), whereas Sm-C supported the simultaneous biosynthesis of both heparan and dermatan sulfate (2.5- and 1.8-fold increments, respectively). Moreover, Sm-C proved capable of diverting FSH-driven proteoglycan biosynthesis from the exclusive stimulation of dermatan sulfate towards the enhanced production of heparan sulfate over dermatan sulfate. These findings suggest that while Sm-C may synergize with FSH in stimulating granulosa cell proteoglycan biosynthesis, it is also able to act in tis own right to effect marked quantitative as well as qualitative alterations in proteoglycan economy. Given the possible role of proteoglycans in follicular antrum formation and follicular atresia, our findings raise the possibility that Sm-C of granulosa cell origin may partake in the growth as well as the demise of the developing ovarian follicle.     

Proteoglycans and hypertension: III: Aorta proteoglycans in Dahl salt-sensitive hypertensive rats

The vascular proteoglycans probably have an important influence on the biomechanical properties of blood vessels and, therefore, may play a role in the development or maintenance of hypertension. In the aorta of the spontaneously hypertensive rat, the authors previously observed an increased content of chondroitin sulfate, an increased incorporation of [35S]sulfate into proteoglycans, and qualitative alterations in the [35S]polysaccharides compared to the normotensive Wistar Kyoto rat. To determine if these differences were related to hypertension or to strain variations, normotensive and hypertensive Dahl S rats were studied. There was a significant elevation (70%) in the aorta content of chondroitin sulfate, whereas the dermatan sulfate and hyaluronic acid contents were similar in the two groups. The in vitro incorporation of [35S]sulfate was increased 2.6-fold in the hypertensive animals. No differences between the two groups were observed with respect to the gel chromatographic profiles of the [35S]proteoglycans or the charge density of the [35S]glycosaminoglycans, as assessed by ion exchange chromatography. It was concluded that the increase in chondroitin sulfate and [35S]sulfate incorporation into proteoglycans occurred as a result of hypertension, regardless of genetic factors.     

Varied low density lipoprotein binding property of proteoglycans synthesized by vascular smooth muscle cells cultured on extracellular matrix

Earlier we showed that the extracellular matrix (ECM) secreted by vascular cells modulated proteoglycan synthesis by vascular smooth muscle cells in culture and altered the proteoglycan characteristics. In this study, we tested the hypothesis that these ECM-mediated alterations increased the affinity of the proteoglycans for plasma low density lipoprotein (LDL). Newly synthesized proteoglycans were isolated from smooth muscle cells cultured on the ECMs secreted by vascular endothelial cells, smooth muscle cells, or THP-1 macrophages and their binding affinity for LDL determined. Proteoglycans from all cultures contained sub-fractions that bound LDL with low and high affinity. However, compared with the cells plated on the endothelial cell ECM, the cells plated on the smooth muscle cell ECM and macrophage ECM synthesized significantly more high affinity proteoglycans. Removal of collagen, elastin, and chondroitin sulfates from the smooth muscle cell ECM and chondroitin sulfates from the macrophage ECM increased the production of high affinity proteoglycans by 15-22%. However, neutralization of fibronectin from both ECMs decreased the high affinity proteoglycans by 20%. Removal of matrix-bound growth factors had no effect on the synthesis of high affinity proteoglycans. Compared with the low affinity proteoglycans, the high affinity proteoglycans were larger, more sulfated and contained higher proportions of chondritin sulfate, dermatan sulfate, and N-sulfated heparan sulfate chains. These results suggest that the ECM-mediated alterations in vascular smooth muscle cell proteoglycans may lead to increased deposition of LDL in the arterial wall.     

Effect of growth factors on hyaluronan and proteoglycan synthesis by retroocular tissue fibroblasts of Graves' ophthalmopathy in culture.

One of the pathological changes seen in Graves' ophthalmopathy is the deposition of glycosaminoglycans such as hyaluronan and proteoglycan in retroocular connective tissue. We analyzed glycosaminoglycans synthesized by retroocular tissue fibroblasts in culture derived from an individual not suffering from thyroid disease and from three patients with Graves' ophthalmopathy. Retroocular tissue fibroblasts synthesized both hyaluronan and proteoglycan, the latter composed mainly of chondroitin sulfate. This contrasts with the proteoglycan synthesized by adult skin fibroblasts which was composed of dermatan sulfate and heparan sulfate proteoglycan. Chondroitin sulfate proteoglycan secreted by retroocular tissue fibroblasts consisted of large and small chondroitin sulfate proteoglycans (CS-PG), their size being determined by the Sepharose CL-6B column. The effects of IGF-1 and PDGF on hyaluronan and proteoglycan synthesis were studied separately and in combination. Both IGF-1 and PDGF increased the synthesis of hyaluronan and proteoglycan in a dose-dependent manner. IGF-1 predominantly stimulated secretion of small CS-PG, while PDGF increased large CS-PG markedly when studied in retroocular tissue fibroblasts. In contrast, IGF-1 stimulated secretion of small proteoglycan while PDGF had little effect on proteoglycan synthesis in skin fibroblasts. Thus, glycosaminoglycan synthesized by retroocular tissue fibroblasts has a unique composition and each component is regulated independently, at least in part.     

The synthesis of glycosaminoglycans in isolated hepatocytes during experimental liver fibrogenesis

During human and experimental liver fibrogenesis, the pattern of glycosaminoglycans in fibrotic liver matrix is greatly changed by severalfold increases of hyaluronic acid, chondroitin sulfate, and dermatan sulfate, respectively. The present study aimed to determine whether hepatocytes take part during fibrogenesis in the alteration of the glycosaminoglycan profile in liver matrix. Rats received thioacetamide orally for 2 and 10 weeks, respectively. After 10 weeks a typical micronodular cirrhosis had developed. Hepatocytes isolated at these time points were characterized by light and electron microscopy and incubated for up to 4 h in suspension cultures in [35S]-sulfate and [3H]-glucosamine containing medium to study the synthesis and intra-/extracellular distribution of total and specific types of glycosaminoglycans. A biphasic change of glycosaminoglycan synthesis in hepatocytes was found. After 2 weeks of TAA-treatment parenchymal cells synthesized about 25% more labeled glycosaminoglycans than control liver cells, but at 10 weeks the synthesis was reduced by more than 40%. Thus, between 2 and 10 weeks of TAA-treatment hepatocellular glycosaminoglycan synthesis decreased by more than 50%. The major portion of newly synthesized glycosaminoglycans was nitrous acid labile and, hence, identified as heparan sulfate. Its fractional synthesis decreased from 0.90 in control cells to 0.84 (2 weeks TAA) and 0.76 (10 weeks TAA), respectively. Thus, the absolute synthesis of heparan sulfate was reduced by 50% in hepatocytes from cirrhosis liver. Eighty to 90% of labeled glycosaminoglycans remained cell-associated. Hyaluronic acid was detected neither in normal hepatocytes nor in hepatocytes from injured liver. We conclude from these data that parenchymal liver cells will not contribute actively to the accumulation of galactosaminoglycans (chondroitin sulfate, dermatan sulfate) and hyaluronic acid in the extracellular matrix during fibrogenesis. The diminished rate of synthesis of heparan sulfate in hepatocytes from cirrhotic liver might explain its fractional decrease in cirrhotic liver matrix.     

Effect of hypoxia and hypoxia/reoxygenation on proteoglycan metabolism by vascular smooth muscle cells

Hypoxia and hypoxia/reoxygenation are known to affect vascular smooth muscle cell physiology. In this study, we first investigated proteoglycan synthesis by human aortic smooth muscle cells exposed to normoxia, hypoxia, or hypoxia/reoxygenation. We then compared the newly synthesized proteoglycans from normoxic and hypoxic-reoxygenation cultures for their ability to bind low density lipoprotein (LDL). Confluent smooth muscle cells under normoxia, hypoxia, or hypoxia/reoxygenation were pulsed with [35S]sulfate, and secreted and cell-associated proteoglycans were analyzed. Secreted proteoglycans in cultures exposed to hypoxia (4 h)/reoxygenation (19 h) increased 28% over those of cells continuously exposed to normoxia. Cell-associated proteoglycans did not differ significantly between the two groups. In contrast, hypoxia (4 h) followed by a 30-min reoxygenation produced a 37% decrease in newly synthesized proteoglycans. Hypoxia alone also resulted in a 24% decrease in secreted proteoglycans and a 20% decrease in cell-associated proteoglycans. Proteoglycans newly synthesized by smooth muscle cells exposed to normoxia and hypoxia/reoxygenation did not differ in their charge densities and molecular size but did differ in glycosaminoglycan composition. Exposure of smooth muscle cells to hypoxia/reoxygenation produced a 60% increase in a proteoglycan subfraction that bound LDL with very high affinity. The incorporation of [3H]leucine into total cellular protein decreased significantly following exposure of smooth muscle cells to hypoxia as well as hypoxia/reoxygenation. These results indicate that hypoxia and hypoxia/reoxygenation cause major alterations in proteoglycan metabolism by vascular smooth muscle cells.     

Macrophage plasma membrane chondroitin sulfate proteoglycan binds oxidized low-density lipoprotein

Lipoprotein interactions with macrophage proteoglycans (PGs) is believed to play an important role in the cellular uptake of lipoproteins and in macrophage cholesterol accumulation. Recently, we have shown the participation of macrophage plasma membrane glycosaminoglycans (GAGs) in the cellular uptake of oxidized LDL (Ox-LDL). The aim of the present study was to identify the specific cell surface proteoglycans involved in this interaction. J-774 A.1 macrophage-like cell line plasma membrane proteoglycans were isolated by anion exchange chromatography from cells that were prelabeled with [35S]sodium sulfate. Using Sepharose 6B chromatography, cell surface major proteoglycans were identified as chondroitin sulfate (CS) proteoglycans (77%) and heparan sulfate (HS) proteoglycans (23%). Binding rates of these 35S-labeled proteoglycans to Ox-LDL and to native LDL were analyzed by their ability to bind lipoproteins coupled to a CnBr-activated Sepharose CL-4B chromatography. Of the total labeled cell surface proteoglycans added to the column, 57% were bound to the Sepharose-coupled Ox-LDL, whereas 73% of the cell surface proteoglycans were bound to the Sepharose-coupled native LDL. Binding of the plasma membrane macrophage 35S-labeled proteoglycans to Ox-LDL was inhibited by adding increasing concentrations of non-labeled chondroitin sulfate, or by pretreatment of the 35S-labeled proteoglycans fraction with chondroitinase ABC. In contrast, neither the addition of non-labeled heparan sulfate, nor pretreatment of the labeled proteoglycans fraction with heparinase III, had any significant effect on proteoglycan binding to Ox-LDL. These findings were further supported by using mutant cells characterized by specific glycosaminoglycan deficiencies. Ox-LDL binding and degradation by mutant 745 CHO cells which are characterized by a deficiency in both heparan sulfate and chondroitin sulfate, was decreased by 28 and 27% respectively, compared to the binding of Ox-LDL to the wild-type CHO cells. Ox-LDL binding and degradation by mutant 677 CHO cells, which lack heparan sulfate but have increased levels of chondroitin sulfate, however, was found to be increased by 29 and 19%, respectively, compared to Ox-LDL binding to the wild-type CHO cells. Finally, analysis of the cell surface proteoglycans in macrophages that were subjected to oxidative stress, by their preincubation with angiotensin II, exhibited a 51-59% increase in their cell surface proteoglycan content, with a major effect on chondroitin sulfate proteoglycans. The present study thus demonstrated that Ox-LDL can specifically bind to macrophage surface chondroitin sulfate proteoglycans, and the macrophage content of this proteoglycan is increased under oxidative stress. The interaction between macrophage chondroitin sulfate proteoglycans and Ox-LDL can contribute to enhanced uptake of Ox-LDL with the formation of cholesterol-loaded foam cells, and accelerated atherosclerosis.     

The effect of transforming growth factor-beta 1 on glycosaminoglycan production by human marrow cultures

We have assayed the effect of transforming growth factor-beta 1 (TGF-beta 1), a potent modulator of hematopoiesis, on glycosaminoglycan production in human marrow cultures. Glycosaminoglycans are a component of the extracellular matrix known to affect cell growth and differentiation. TGF-beta 1 and [35S]sulfate were added simultaneously to hematopoietically active human marrow cultures, and radiolabeled glycosaminoglycan production was determined by cetylpyridinium chloride precipitation. TGF-beta 1 at 15 ng/ml for 72 h increased [35S]sulfate incorporation into media glycosaminoglycans to 190% of control levels but did not affect the [35S]sulfate incorporation into cell-associated glycosaminoglycans. Approximately 90% of the glycosaminoglycans in the media fraction and 85% of the glycosaminoglycans in the cell-associated fraction were susceptible to degradation by chondroitin ABCase in both treated and control cultures. Pulse-chase experiments suggested that the increase in glycosaminoglycan [35S]sulfate incorporation was not due to decreased glycosaminoglycan degradation. This concentration of TGF-beta 1 did not alter nonadherent granulocyte-macrophage colony-forming unit (CFU-GM) number per flask but significantly decreased the more primitive adherent CFU-GM number per flask (by 50%-70%). These data suggest that the ability of TGF-beta 1 to modulate hematopoiesis may be due, in part, to its effects on glycosaminoglycan production.     

Formation of extracellular matrix in normal rat liver: lipocytes as a major source of proteoglycan

Proteoglycans are a major component of the normal hepatic extracellular matrix and undergo quantitative and qualitative changes in hepatic fibrosis. The cellular sources of proteoglycans are as yet incompletely defined. We examined this question using primary cultures of hepatocytes and lipocytes isolated from normal rat liver. Proteoglycan synthesis was assessed by measuring production of sulfated glycosaminoglycan, the polysaccharide moiety of proteoglycans. The findings indicate that lipocytes produce sixfold more glycosaminoglycan, per cell, than do hepatocytes. Two-thirds of the newly synthesized material is cell- or matrix-associated. Of the individual glycosaminoglycan species produced by lipocytes, dermatan sulfate represents 60% of the total; heparan sulfate and chondroitin sulfate are measurable but relatively minor. In hepatocyte cultures, heparan sulfate accounted for essentially all of the glycosaminoglycan detected. We conclude that lipocytes are an important source of proteoglycan in normal liver and may be the principal source of dermatan sulfate associated with hepatic fibrosis.     

Age-related changes in the plasma membrane proteoglycans of rat kidney glomerular cells

In a previous in vivo study, we showed that the glomerular cells of rat kidney synthesize both peripheral and integral plasma membrane proteoglycans. The present work focusses on the age-related changes in these cell membrane proteoglycans. The peripheral proteoglycans in “adult control” rats aged 3 months were found to be heparan sulfate, dermatan sulfate, and chondroitin sulfate, with heparan sulfate being the main glycosaminoglycan. The integral membrane proteoglycans contained mainly dermatan sulfate plus less amounts of heparan sulfate. The relative proportions of the glycosaminoglycans in the integral membrane proteoglycans changed between 1 and 3 months. In addition, the degree of sulfation increased in both families of proteoglycans, and this was associated with an increase in glycosaminoglycan synthesis in the peripheral proteoglycans. The nature and relative proportions of the glycosaminoglycans forming the proteoglycans, did not change with age, after 10 months, and neither did the amount of glycosaminoglycans. But, the degree of sulfation of both peripheral and integral membrane proteoglycans decreased. De novo synthesized proteoglycans from 24-month-old rats had a higher overall charge than did those at other ages, owing to the presence of sulfate and carboxylic groups. We conclude that, as for glomerular basement membrane proteoglycans, biochemical alterations affect the glomerular cell membrane proteoglycans with aging.     

Thyroid hormone excess stimulates the synthesis of proteoglycan in human skin fibroblasts in culture

We previously demonstrated that proteoglycan accumulated in the affected skin of circumscribed pretibial myxedema of Graves' disease. As an underlying mechanism responsible for the accumulation, we sought to determine whether excess thyroid hormone was partially responsible for the increase in proteoglycan synthesis. Human skin fibroblasts were cultured in Ham's F-10 medium containing 1% Nutridoma with graded doses of T3 (0.184 x 10(-9) to 46 x 10(-9) mol/l) and were labelled with [35S]sulphate and [3H]glucosamine. Proteoglycans were purified by Sephadex G-50, Q-Sepharose chromatography with NaCl-gradient and Sepharose CL-6B chromatography. 35S and 3H incorporated into dermatan sulphate proteoglycan and heparan sulphate proteoglycan and 3H incorporated into hyaluronan were measured. 35S and 3H incorporation into dermatan sulphate proteoglycan was minimum at a T3 concentration of 0.184 x 10(-9) mol/l, and increased with increasing doses of T3 up to 46 x 10(-9) mol/l. 35S and 3H incorporation into heparan sulphate proteoglycan also increased with increasing-doses of T3. 3H incorporation into hyaluronan was not influenced at all by T3. The increased incorporation of 35S into proteoglycan in high-T3 culture reflects the increased synthesis of proteoglycan because 1. the extent of sulphation of disaccharides examined by thin-layer chromatography was not altered by T3; 2. the specific activity of [35S]sulphate was not influenced by T3, and 3. T3 did not decrease the degradation rate of cell-associated proteoglycan.