Effect of retinoic acid on in vitro proliferation activity and glycosaminoglycan synthesis of mesenchymal cells from palatal shelves of mouse fetuses

To clarify the mechanism by which retinoid causes cleft palate, we investigated the effect of retinoic acid (RA) on proliferation activity and glycosaminoglycan (GAG) synthesis in mouse fetuses palatal mesenchymal (MFPM) cells. MFPM cells were incubated for 1-11 days with various concentrations of RA to examine its effect on growth rate. Also, confluent cultures were incubated with [3H]glucosamine or [35S]sulfate in the presence of various concentrations of RA to investigate the effect of RA on GAG synthesis. RA remarkably inhibited the growth of MFPM cells in a dose-dependent manner. RA also inhibited the synthesis of GAGs, with sulfated GAGs being more severely affected than hyaluronic acid. These data suggest that the inhibition of proliferation activity and GAG synthesis of palatal mesenchymal cells might be involved in the induction of cleft palate by retinoic acid.     

Effect of glucocorticoid on glycosaminoglycans synthesis in cultured mouse embryonic palatal mesenchymal cells

The effect of glucocorticoids (GC) on the synthesis of glycosaminoglycans (GAGs) in mesenchymal cells obtained from palatal shelves of mouse embryos was studied. Both hyaluronic acid (HA) synthesis and sulfated GAG synthesis were inhibited with triamcinolone acetonide (TA) in a concentration-dependent manner. However, the degree of inhibition of synthesis with TA was greater for HA than for the sulfated GAGs. To determine whether the inhibitory effects of TA on HA and sulfated GAGs synthesis were mediated through GC receptors, we performed experiments using progesterone, vitamin B6, and molybdate. The inhibitory effect of TA was antagonized by all three. The results obtained in the present study suggest that the inhibition of synthesis of HA and sulfated GAGs with GC is mediated through GC receptors and is involved in cleft palate induction by GC.     

Epidermal growth factor potentiates the induction of ornithine decarboxylase activity by prostaglandins in embryonic palate mesenchymal cells: effects on cell proliferation and glycosaminoglycan synthesis

Epidermal growth factor (EGF) and prostaglandins (PGs) have been implicated in the regulation of a number of developmental processes in the mammalian embryonic palate. Normal palatal ontogenesis is dependent on the presence and quite possibly on the interaction of various hormones and growth factors. The interaction between EGF and PGs in regulation of murine embryonic palate mesenchymal (MEPM) cell growth and differentiation was therefore investigated by monitoring the activity of ornithine decarboxylase (ODC), the principle and rate limiting enzyme of polyamine biosynthesis. ODC activity is tightly coupled to the proliferative and differentiative state of eukaryotic cells and therefore serves as a reliable indicator of such cellular functions. Treatment of confluent cultures of MEPM cells with EGF (1-50 ng/ml) resulted in a dose-related increase in ODC activity, while similar treatment with either PGE2 or PGF2 alpha (at concentrations up to 1 microM) did not elicit a dose-dependent increase in enzyme activity. Concurrent treatment of MEPM cells with EGF (20 ng/ml) and either PGE2 or PGF2 alpha (0.1-10000 nM) resulted in a marked prostaglandin dose-dependent induction of ODC activity, suggesting a strong cooperative interaction between these factors. ODC activity was maximal by 4 to 8 hr and could be completely inhibited by preincubation of the cells with actinomycin D or cycloheximide, indicating that de novo synthesis of RNA and protein is necessary for enzyme induction. Stimulation of ODC activity by EGF and PGE2 in these cells was not positively correlated with the level of cellular DNA synthesis but did result in a ninefold increase in the synthesis of extracellular glycosaminoglycans (GAGs), a key macromolecular family implicated in palatal morphogenesis. Stimulation of GAG synthesis was significantly inhibited by the administration of 5 mM DFMO (an irreversible inhibitor of ODC), indicating that the marked increase in GAG production was dependent, in part, on the induction of ODC activity by EGF and PGE2. Qualitative analysis of the palatal GAGs indicated that synthesis of several major classes of GAGs was stimulated. Collectively these data demonstrate a cooperative interaction between EGF and PGs in the induction of ODC activity. Such activity may serve to regulate the synthesis of GAGs, which are instrumental in mammalian palatal ontogenesis.     

Glucose deficiency inhibits glycosaminoglycans synthesis in fibroblast cultures

We decided to study the effect of glucose deprivation on glycosaminoglycan (GAG) synthesis and degradation in fibroblast cultures, vitality of these cells and a correlation of these processes with the expression of oxygen/glucose-regulated proteins (ORP150/GRP170). The incorporation of [³H]-glucosamine into both newly synthesised hyaluronic acid and sulphated GAGs and [³⁵S]-sulphate into GAGs was used as an index of glycosaminoglycan synthesis. Quantitative evaluation of newly synthesised GAGs degradation was determined by pulse-chase experiments. We demonstrated that fibroblasts incubated in high glucose medium synthesised significant amounts of GAGs. Most of them were secreted into the culture medium. The shortage of glucose resulted in about 40% reduction in synthesis of GAGs, both those secreted into culture medium and remaining in the cell layer. The pulse-chase experiments demonstrated that the reduced amount of newly synthesised glycosaminoglycans was protected against intracellular degradation. Proportionally less GAGs were degraded in cultures incubated in low glucose than in high glucose media. These phenomena were accompanied by an increase in the expression of chaperon – ORP150 in cultures growing in low glucose medium. We suggest that the increased expression of ORP150 is a factor which prolongs the cell vitality and protects glycosaminoglycans against intracellular degradation induced by glucose deprivation.     

Effects of thyroid-stimulating hormone and phorbol ester on glycosaminoglycan synthesis in porcine thyroid epithelial cells in primary culture

The effects of thyroid-stimulating hormone (TSH) and a tumor promoter: 12-0-tetradecanoyl-phorbol-13-acetate on glycosaminoglycan (GAG) synthesis were studied in porcine thyroid epithelial cells in primary culture. TSH is known to involve cyclic AMP mechanism and phorbol ester to act by protein kinase C pathway. Chronic treatment of cells with TSH increased the synthesis of heparan sulphate associated with the cell layer and hyaluronic acid in the culture medium. Phorbol ester increased the radioactivity of total GAGs in the culture medium but had no effect on GAGs associated with the cell layer. It inhibited the positive effect of TSH on heparan sulphate synthesis. These results suggest that in thyroid epithelial cells the synthesis of the GAGs associated with the cell layer and those secreted into the culture medium are regulated by different intracellular mechanisms.     

Transforming growth factor-beta modulation of glycosaminoglycan production by mesenchymal cells of the developing murine secondary palate

Development of the mammalian secondary palate requires proper production of the extracellular matrix, particularly glycosaminoglycans (GAGs) and collagen. Endogenous factors that regulate the metabolism of these molecules are largely undefined. A candidate for a locally derived molecule would be transforming growth factor beta 1 (TGF beta 1) by virtue of its potency as a modulator of extracellular matrix metabolism by several cell lines. We have thus attempted to assign a regulatory role for TGF beta 1 in modulation of GAG production and degradation by mesenchymal cells of the murine embryonic palate (MEPM). Treatment with TGF beta 1 or TGF beta 2, but not IGF-II, resulted in a stimulation of total GAG synthesis. Furthermore, cells treated with both TGF beta 1 and TGF alpha showed a synergistic increase in GAG synthesis if pretreated with TGF beta 1 but not TGF alpha. Simultaneous stimulation with TGF beta 1 and TGF beta 2 did not elicit a synergistic response. These studies demonstrate the ability of TGF beta, synthesized by embryonic palatal cells, to specifically stimulate GAG synthesis by MEPM cells. Other growth factors present in the developing craniofacial region may also modulate TGF beta-induced GAG synthesis, a biosynthetic process critical to normal development of the embryonic palate.     

Turnover of sulfated glycosaminoglycans in fibroblasts derived from patients with Werner''s syndrome

Fibroblasts derived from patients with Werner's syndrome (WS) were incubated with radioactive sulfate to study the incorporation of 35S into glycosaminoglycans (GAGs). The accumulation of cell-associated 35S radioactivity in the GAGs of WS fibroblasts was consistently higher than parallel accumulation in normal human fibroblasts, but was substantially less than in fibroblasts derived from patients with Hurler's syndrome (HS). However, when fibroblasts were labeled with 35SO4(2-), trypsinized to remove extracellular and pericellular radioactive GAGs, replated, and chased to follow the fate of the intracellular radioactivity, both WS and normal cells showed a rapid release of the intracellular 35S, while HS cells showed little or no loss of intracellular radioactivity. The radioactivity released from WS and normal cells was of low molecular weight (LMW), eluting from gel filtration columns at the same position as free sulfate. These results establish that WS cells degrade intracellular sulfated GAGs and argue against the hypothesis that a defect in GAG degradation pathways is the basis for the increased level of cell-associated GAGs. Other possible explanations for the increased cell-associated [35S]GAGs in WS cells as compared with normal cells were also considered: increased GAG sulfation; an increase in GAG chain length; an increased rate of GAG synthesis; and a decreased rate of shedding of cell surface proteoglycan into the medium. No difference between normal and WS fibroblasts in any of the above parameters was observed. These results strongly imply that the primary biochemical defect in WS fibroblasts does not involve sulfated GAG metabolism.     

Effects of 4-deoxy-L-threo-pentose, a novel carbohydrate, on neural cell proteoglycan synthesis and function.

A novel carbohydrate, 4-deoxy-L-threo-pentose (4-deoxyxylose), was synthesized by way of reductive dechlorination of a chlorodeoxy sugar. This carbohydrate, an analogue of xylose which is required for the initiation of glycosaminoglycan (GAG) synthesis, was used to explore the function of GAG side chains in neurite outgrowth on a laminin substrate. 4-Deoxyxylose inhibited the incorporation of 35SO4 into the GAGs of neuronal and astrocytic proteoglycans, with no effect being seen on the incorporation of [3H]glucosamine into proteoglycan. Direct analysis of the heparan sulphate fraction from such cells using nitrous acid digestion confirmed that the GAGs were undersulphated. No inhibition of either 35SO4 or [3H]glucosamine incorporation was observed in primary mouse hepatocytes exposed to 4-deoxyxylose. 4-Deoxyxylose produced a direct dose-dependent inhibition of neurite outgrowth by sensory neurons, and medium conditioned by neurons or astrocytes in the presence of 4-deoxyxylose displayed less laminin-complexed neurite-promoting activity than medium conditioned in its absence. These data suggest that 4-deoxyxylose inhibits neurite outgrowth by altering the sulphation of the GAGs of heparan sulphate proteoglycans.     

Albumin Synthesis by Rat Hepatocytes Cultured on Collagen Gels Is Sustained Specifically by Heparin

We investigated the influence of various kinds of glycosaminoglycans (GAGs) in collagen gels on the maintenance of albumin synthesis in primary culture of rat hepatocytes. Among the GAGs examined (heparin, heparan sulfate, keratan sulfate, chondroitin sulfate A, dermatan sulfate, and hyaluronic acid), only heparin-containing collagen gel cultures could significantly sustain albumin synthesis. However, other GAGs, such as heparan sulfate and keratan sulfate, had almost no effect on the maintenance of albumin synthesis. Heparin in collagen gels exhibited a dose-dependent effect on albumin synthesis: heparin at 400 μg/ml-collagen solution maintained albumin synthesis for over 3 weeks. On the other hand, when an equivalent amount of heparin was added directly to the collagen gel culture medium, it prolonged albumin synthesis for only 10 days. The results demonstrate that specific regulation of albumin synthesis by heparin was significantly promoted by coincubating it with collagen, suggesting that some specific interaction between heparin and collagen might be of importance for the maintenance of hepatocyte functions.     

Effects of chlorcyclizine-induced glycosaminoglycan alterations on patterns of hyaluronate distribution during morphogenesis of the mouse secondary palate

Chlorcyclizine (CHLR) enhances the degradation of hyaluronate (HA) into smaller molecular weight pieces with no effect on its synthesis. Administration of CHLR to pregnant CD-1 mice on gestational days 10.5, 11.5 and 12.5 results in 100% cleft palate in the fetuses. The caudal two thirds of the palatal shelves are reduced in size and unable to reorient in vitro, while anterior shelf regions are relatively unaffected. Alcian blue staining combined with specific enzymic digestion was used to identify HA in sections of CHLR-treated shelves. With the aid of computer-assisted image subtraction the patterns of HA distribution across the tissue section were objectively identified. Anterior, posterior and presumptive soft palatal shelf regions were examined at gestational days 13.25, 13.5, 13.75 and 14.5. Acquisition of a normal pattern of HA distribution was delayed by about 24 h, as compared to untreated specimens in all three shelf regions. The posterior and soft regions, comprising the caudal two thirds of the shelf, also showed pronounced shape change. These regions only displayed normal curvature of the nasal surface when a normal pattern of HA distribution was attained. These results suggest that, for the caudal two thirds of the palatal shelf, normal shape and the ability to remodel are linked to the molecular configuration of HA and to a specific pattern of HA distribution.     

Inhibition of palatal epithelial cell death by altered protein synthesis.

Programmed cell death occurs in a defined region of the secondary palatal epithelium (medial edge) during its development in vivo or in vitro. The purpose of this study was to examine the effects of various inhibitors of macromolecular synthesis, particularly glycoprotein synthesis, on the death of palatal medial-edge epithelial cells in vitro. Rat palatal shelves explanted on gestational Day 15 and cultured singly showed medial-edge cell degeneration after 48 hr, whereas in the presence of 6-diazo-5-oxo-l-norleucine (DON), 2-deoxyglucose (DOG), or cycloheximide, cell death was prevented. This was in contrast to the lack of inhibition of cell death in the presence of actionomycin D or cytosine arabinoside. Selectively adding metabolites which bypass the effect of DON on various synthetic pathways demonstrated that the inhibition of cell death by DON was most likely due to a block in glucosamine formation. The synthesis and activity of various lysosomal enzymes were not affected by DON, whereas a marked decrease was observed with both cycloheximide and DOG. These results suggest that palatal epithelial cell death is not a passive event, but an active process requiring the programmed synthesis of specific proteins.     

Alterations of membrane lipids and in gene expression of ganglioside metabolism in different brain structures in a mouse model of mucopolysaccharidosis type I (MPS I)

Mucopolysaccharidosis I (MPS I) is a congenital disorder caused by the deficiency of α-l-iduronidase (IDUA), with the accumulation of glycosaminoglycans (GAGs) in the CNS. Although GAG toxicity is not fully understood, previous works suggest a GAG-induced alteration in neuronal membrane composition. This study is aimed to evaluate the levels and distribution of gangliosides and cholesterol in different brain regions (cortex, cerebellum, hippocampus and hypothalamus) in a model using IDUA knockout (KO) mice (C57BL/6). Lipids were extracted with chloroform–methanol and then total gangliosides and cholesterol were determined, followed by ganglioside profile analyses. While no changes in cholesterol content were observed, the results showed a tissue dependent ganglioside alteration in KO mice: a total ganglioside increase in cortex and cerebellum, and a selective presence of GM3, GM2 and GD3 gangliosides in the hippocampus and hypothalamus. To elucidate this, we evaluated gene expression of ganglioside synthesis (GM3, GD3 and GM2/GD2 synthases) and degradation of (Neuraminidase1) enzymes in the cerebellum and hippocampus by RT-sq-PCR. The results obtained with KO mice showed a reduced expression of GD3 and GM2/GD2 synthases and Neuraminidase1 in cerebellum; and a decrease in GM2/GD2 synthase and Neuraminidase1 in the hippocampus. These data suggest that the observed ganglioside changes result from a combined effect of GAGs on ganglioside biosynthesis and degradation.     

Genistein-mediated inhibition of glycosaminoglycan synthesis, which corrects storage in cells of patients suffering from mucopolysaccharidoses, acts by influencing an epidermal growth factor-dependent pathway

Background  

Mucopolysaccharidoses (MPS) are inherited metabolic disorders caused by mutations leading to dysfunction of one of enzymes involved in degradation of glycosaminoglycans (GAGs). Due to their impaired degradation, GAGs accumulate in cells of patients, which results in dysfunction of tissues and organs. Substrate reduction therapy is one of potential treatment of these diseases. It was demonstrated previously that genistein (4', 5, 7-trihydroxyisoflavone) inhibits synthesis and reduces levels of GAGs in cultures of fibroblasts of MPS patients. Recent pilot clinical study indicated that such a therapy may be effective in MPS III (Sanfilippo syndrome).     

Nerve dependent sulphated glycosaminoglycan synthesis in limb regeneration of the newt Pleurodeles waltl

Denervation of the amputated limb of newts stops the regeneration process by decreasing blastema cell proliferation. We investigated the effect of the denervation on each of the two compartments (epidermal cap, mesenchyme) in mid-bud blastemas on the level of sulphated glycosaminoglycans (GAGS). Denervation resulted in an increase of about threefold in the incorporation of [³⁵S] sulphate into mesenchyme GAGs but had no effect on the epidermal cap. The increase of GAG synthesis in the mesenchymal part of the blastema involved both heparan sulphates and chondroitin-dermatan sulphates. Gel filtration showed no change in GAGs size after denervation. These results confirm that the mesenchymal part of the mid-bud blastema is the main target of nerves and, as heparan sulphates are known to store acidic fibroblast growth factor (aFGF), a polypeptide found in the blastema (Boilly et al.. 1991), this suggest that the nerves' effect on glycosaminoglycans turnover could be implicated in the control of bioavailability of this growth factor in the blastema.     

Follicular fluid glycosaminoglycans inhibit degradation of low-density lipoproteins and progesterone production by porcine granulosa cells

Porcine granulosa cells were incubated with commercially available glycosaminoglycans (GAGs) or GAGs purified from porcine follicular fluid to evaluate the effects of GAGs on degradation of low-density lipoproteins (LDL) and progesterone production. Commercially available heparin and chondroitin sulfates (CS) as well as follicular CS and heparan sulfate (HS) inhibited degradation of LDL in dose-dependent manners. Doses of follicular CS and HS required to inhibit 50% of the LDL degradation corresponded to concentrations found in follicular fluid (less than 1 mg/ml). Progesterone production was also inhibited in a dose-dependent fashion by follicular GAGs at concentrations found in follicles. The ability of the follicular GAGs to inhibit degradation of LDL could represent a mechanism by which the utilization of LDL-derived sterol is temporarily restricted following permeabilization of the ovulatory follicle. Follicular GAGs may also modulate utilization of apoprotein E-containing high-density lipoproteins in unruptured follicles.     

Dexamethasone affects phosphatidylinositol synthesis and degradation in cultured human embryonic cells

Dexamethasone (DEX), a glucocorticoid which induces cleft palate, causes marked alterations in the synthesis and degradation of phosphatidylinositol (PI) but not phosphatidylcholine in an established fibroblastic cell line derived from a human embryonic palate. Incorporation of radiolabeled inositol into phosphatidylinositol as well as degradation of prelabeled phosphatidylinositol is stimulated by DEX. The dose-response curves for the DEX-induced effect on PI synthesis and DEX-induced inhibition of cell proliferation are nearly identical, with the maximal responses occurring at 10^?8M DEX. Our results suggest that DEX-induced inhibition of human embryonic palatal mesenchyme cell proliferation and alterations in synthesis and degradation of phosphatidylinositol are related.     

Regulation of collagenase activities of human cathepsins by glycosaminoglycans

Cathepsin K, a lysosomal papain-like cysteine protease, forms collagenolytically highly active complexes with chondroitin sulfate and represents the most potent mammalian collagenase. Here we demonstrate that complex formation with glycosaminoglycans (GAGs) is unique for cathepsin K among human papain-like cysteine proteases and that different GAGs compete for the binding to cathepsin K. GAGs predominantly expressed in bone and cartilage, such as chondroitin and keratan sulfates, enhance the collagenolytic activity of cathepsin K, whereas dermatan, heparan sulfate, and heparin selectively inhibit this activity. Moreover, GAGs potently inhibit the collagenase activity of other cysteine proteases such as cathepsins L and S at 37 degrees C. Along this line MMP1-generated collagen fragments in the presence of GAGs are stable against further degradation at 28 degrees C by all cathepsins but cathepsin K, whereas thermal destabilization at 37 degrees C renders the fragments accessible to all cathepsins. These results suggest a novel mechanism for the regulation of matrix protein degradation by GAGs. It further implies that cathepsin K represents the only lysosomal collagenolytic activity under physiologically relevant conditions.     

Inhibition of branching morphogenesis and alteration of glycosaminoglycan biosynthesis in salivary glands treated with β-d-xyloside

The role of glycosaminoglycans (GAGs) in the branching morphogenesis of embryonic mouse salivary glands was investigated by culturing the glands in the presence of xylose derivatives which stimulate synthesis of the xyloselinked classes of GAGs. Branching morphogenesis is inhibited severely, but reversibly, by 0.5–1.0 mM π-nitrophenyl-β-d-xylopyranoside and the inhibition correlates with a stimulation of incorporation of [³H]glucosamine (1.8-fold) and [³⁵S]sulfate (almost 3-fold) into GAGs. The effect of β-xyloside on accumulation of newly synthesized GAG also occurs in the presence of the protein synthesis inhibitor cycloheximide, suggesting that the production of free GAG chains rather than proteoglycan-associated GAGs is being stimulated. The xyloside effects apparently do not result from general cytotoxicity of the derivatives, since similar concentrations of the α-anomer do not alter salivary branching or GAG synthesis, the rudiments resume morphogenesis when returned to control medium, and the effect on GAG synthesis is stimulatory rather than inhibitory. The study suggests that GAG biosynthesis plays an important role in salivary development, and that xylosides provide useful probes for characterizing the molecular events controlling branching morphogenesis.     

Hypoxia modifies the effect of PDGF on glycosaminoglycan synthesis by primary human lung cells

Hypoxia, a consequence of interstitial lung diseases, may lead to secondary pulmonary hypertension and pulmonary vascular remodeling. Hypoxia induces activation and proliferation of lung cells and enhances the deposition of extracellular matrix including glycosaminoglycans (GAGs). To elucidate the cell biological mechanisms underlying the development of secondary pulmonary hypertension, we studied the effect of hypoxia on GAG synthesis by human lung cells. GAG synthesis was measured by incorporation of [(3)H]glucosamine; GAGs were isolated, purified, and characterized with GAG-degrading enzymes. Fibroblasts and vascular smooth muscle cells (VSMCs) synthesized hyaluronic acid, heparan sulfate, and chondroitin sulfates, whereas dermatan sulfate was found only in fibroblasts. Hypoxia did not influence the size or charge of the individual GAGs. However, hypoxia inhibited platelet-derived growth factor-induced [(3)H]glucosamine incorporation in secreted GAGs, especially hyaluronic acid, in VSMCs. In contrast, it stimulated GAG secretion, specifically heparan sulfate, by fibroblasts. Our results indicate that hypoxia induces modifications in GAG synthesis by human lung VSMCs and fibroblasts that may be correlated to pathophysiological manifestations in lung diseases causing hypoxia.