Effects of calcitonin and parathyroid hormone on the metabolism of chondrocytes in culture

Rabbit articular chondrocytes in suspension culture synthesize Type II colagen [3α₁(II)] in the absence of extracellular Ca²⁺ and Type Icollagen [2α₁?(I)·α₂] in the complete medium. As a result of pre-treatment in monolayer culture with calcitonin or parathyroid hormone in the complete medium, an influx of Ca²⁺ into the cells occurs. These cells produce mainly Type I collagen when transferred to suspension cultures in the medium devoid of CaCl₂. If added directly to the suspension culture medium containing no CaCl₂, calcitonin stimulates an active efflux of Ca²⁺ from the cells into the medium and leads the cells to synthesize Type I collagen. Under similar conditions, parathyroid hormone does not change the collagen-phenotype.     

Elevated extracellular calcium concentrations induce type X collagen synthesis in chondrocyte cultures

Chondrocytes at different stages of cellular differentiation were isolated from the tarsal element (immature chondrocytes) and zones 2 and 3 (mature chondrocytes) of 12-d chick embryo tibiotarsus. The chondrocytes from the two sources differed in their cell morphologies, growth rate and production of type X collagen. In 24 h, zone 2 and 3 chondrocytes synthesized 800 times more type X collagen than tarsal chondrocytes. The effect of exogenous CaCl2 (5 and 10 mM) on the synthesis of type X collagen by both mature and immature chondrocytes was tested. After a 72-h incubation of zone 2 and 3 chondrocytes with CaCl2 type X collagen increased 8-fold with 5 mM and 10-fold with 10 mM Ca2+. [3H]Proline incorporation into culture medium and matrix macromolecules increased 11 and 32% with 5 and 10 mM CaCl2, respectively. Type II collagen synthesis was not affected by elevated extracellular Ca2+ during this 72-h period. Similar studies with tarsal chondrocytes demonstrated a time- and dose-dependent response to CaCl2 with type X collagen levels reaching a 4-fold and 15-fold increase over controls with 5 and 10 mM Ca2+, respectively, at 48 h. Elevated extracellular Ca2+ had no effect on cell proliferation. These observations offer the first direct evidence of the induction of type X collagen synthesis with elevated extracellular Ca2+.     

Regulation of hormone secretion and cytosolic Ca2+ by extracellular Ca2+ in parathyroid cells and C-cells: role of voltage-sensitive Ca2+ channels

The two dihydropyridine enantiomers, (+)202-791 and (-)202-791, that act as voltage-sensitive Ca2+ channel agonist and antagonist, respectively, were examined for effects on cytosolic Ca2+ concentrations ([Ca2+]i) and on hormones secretion in dispersed bovine parathyroid cells and a rat medullary thyroid carcinoma (rMTC) cell line. In both cell types, small increases in the concentration of extracellular Ca2+ evoked transient followed by sustained increases in [Ca2+]i, as measured with fura-2. Increases in [Ca2+]i obtained by raised extracellular Ca2+ were associated with a stimulation of secretion of calcitonin (CT) and calcitonin gene-related peptide (CGRP) in rMTC cells, but an inhibition of secretion of parathyroid hormone (PTH) in parathyroid cells. The Ca2+ channel agonist (+)202-791 stimulated whereas the antagonist (-)202-791 inhibited both transient and sustained increases in [Ca2+]i induced by extracellular Ca2+ in rMTC cells. Secretion of CT and CGRP was correspondingly enhanced and depressed by (+)202-791 and (-)202-791, respectively. In contrast, neither the agonist nor the antagonist affected [Ca2+]i and PTH secretion in parathyroid cells. Depolarizing concentrations of extracellular K+ increased [Ca2+]i and hormone secretion in rMTC cells and both these responses were potentiated or inhibited by the Ca2+ channel agonist or antagonist, respectively. The results suggest a major role of voltage-sensitive Ca2+ influx in the regulation of cytosolic Ca2+ and hormones secretion in rMTC cells. Parathyroid cells, on the other hand, appear to lack voltage-sensitive Ca2+ influx pathways and regulate PTH secretion by some alternative mechanism.     

Differential effects of parathyroid hormone fragments on collagen gene expression in chondrocytes

The effect of parathyroid hormone (PTH) in vivo after secretion by the parathyroid gland is mediated by bioactive fragments of the molecule. To elucidate their possible role in the regulation of cartilage matrix metabolism, the influence of the amino-terminal (NH2-terminal), the central, and the carboxyl-terminal (COOH-terminal) portion of the PTH on collagen gene expression was studied in a serum free cell culture system of fetal bovine and human chondrocytes. Expression of alpha1 (I), alpha1 (II), alpha1 (III), and alpha1 (X) mRNA was investigated by in situ hybridization and quantified by Northern blot analysis. NH2- terminal and mid-regional fragments containing a core sequence between amino acid residues 28-34 of PTH induced a significant rise in alpha1 (II) mRNA in proliferating chondrocytes. In addition, the COOH-terminal portion (aa 52-84) of the PTH molecule was shown to exert a stimulatory effect on alpha1 (II) and alpha1 (X) mRNA expression in chondrocytes from the hypertrophic zone of bovine epiphyseal cartilage. PTH peptides harboring either the functional domain in the central or COOH-terminal region of PTH can induce cAMP independent Ca2+ signaling in different subsets of chondrocytes as assessed by microfluorometry of Fura-2/AM loaded cells. These results support the hypothesis that different hormonal effects of PTH on cartilage matrix metabolism are exerted by distinct effector domains and depend on the differentiation stage of the target cell.     

Phenotypic differences in subclones and long-term cultures of clonally derived rat bone cell lines

Previous studies with clonally derived populations of cells have shown that cells released from embryonic rat calvaria by enzymatic digestion are heterogeneous with respect to their hormone responsiveness, morphology, and production of matrix components [Aubin JE et al; J. Cell Biol 92:452, 1982]. Several of these clonal populations have been used to study the effects of long-term culture and inter- and intraclonal cell heterogeneity. During continuous subculture, marked changes in collagen synthesis were observed in two clonal populations. Both of these clones were originally responsive to parathyroid hormone (PTH) and synthesized primarily type I collagen with small amounts of type III and V collagens, although one clone (RCJ 3.2) had a fibroblastic morphology whereas the second clone (RCB 2.2) displayed a more polygonal shape. Following routine subculture over 3 yr, clone RCB 2.2 was found to synthesize exclusively alpha 1(I)-trimer and not other interstitial collagens. When the same cells were maintained at confluence for 1-2 wk, however, they also synthesized type III collagen. Whereas RCJ 3.2 did not show such dramatic changes in collagen synthesis after long-term subculture, two subclones derived from RCJ 3.2 were found to synthesize almost exclusively either type III collagen (RCJ 3.2.4.1) or type V collagen (RCJ 3.2.4.4). Immunocytochemical staining indicated that both subpopulations also produced type IV collagen, laminin, and basement membrane proteoglycan, proteins that are typically synthesized by epithelial cells. The differences in collagen expression by the various clonal cell populations were accompanied by qualitative and quantitative differences in other secreted proteins and differences in cell morphology. The results demonstrate both the inter- and intraclonal heterogeneity of connective tissue cells and their diverse potentiality with respect to extracellular matrix synthesis.     

Magnesium-dependent attachment and neurite outgrowth by PC12 cells on collagen and laminin substrata

We report a study of the substratum and medium requirements for attachment and neurite outgrowth by cells of the pheochromocytoma-derived PC12 line. In attachment medium containing both Ca2+ and Mg2+, more than 50% of cells attached within 1 hr to petri dishes coated with native collagen Types I/III or II, native or denatured collagen Type IV, laminin, wheat germ agglutinin (WGA), or poly-L-lysine; attachment to dishes coated with nerve growth factor (NGF) was only about 20% and attachment to uncoated dishes or to dishes coated with fibronectin or gelatin was almost nil. Neither prior culturing in the presence of NGF nor addition of NGF to the attachment medium significantly affected the extent of attachment to collagen or laminin. With Ca2+ (1 mM) as the sole divalent cation, cells attached normally to WGA, polylysine, and NGF, but failed to attach to collagen or laminin. With Mg2+ (1 mM) as the only divalent cation, attachment to all substrata was about the same as in medium with both Ca2+ and Mg2+. Like the ionic requirements, the kinetics of attachment, insensitivity to protease treatment of the cells, and inhibition by low temperature and sodium azide were similar for PC12 attachment to collagen and laminin, suggesting that a common molecular mechanism may underlie attachment to these substrata. The only significant difference observed was that addition of WGA (30 micrograms/ml) to the attachment medium inhibited attachment to collagen but promoted attachment to laminin. Finally, PC12 cells extended neurites on laminin, on native collagens I/III, II, and IV, and on denatured collagen IV; they did not extend neurites on denatured collagens I/III or II, NGF, or WGA. Neurite outgrowth on collagen and laminin occurred with Mg2+ as the sole divalent cation. These results suggest that the same Mg2+-dependent adhesion mechanism operates at the cell body and at the growth cone.     

Electrophoretically separated bone cell types from the foetal rat calvarium: A histochemical and biochemical study

Summary Isolated cells obtained from foetal rat bone (calvarium) by collagenase digestion can be separated into three subpopulations on the basis of surface charge by free flow electrophoresis. These subpopulations have been tentatively identified by numerical, biochemical and functional criteria and are believed to be composed of: (1) bone resorbing cell types, designated Peak I cells; (2) fibroblasts and loose connective tissue cells, designated Peak II cells; and (3) a mixture of osteoblasts and osteoprogenitor cell types, designated Peak III cells. the anatomical position of these subpopulations in the whole calvarium was determined by comparing the results of histochemical and morphological experiments with the results of biochemical experiments. It was found that Peak I cells are located predominantly on the ventral (endocranial) surface, Peak II cells in the connective tissue periosteal membranes and Peak III cells on the dorsal (ectocranial) surface and in the suture line areas. The response of these cell types to parathyroid hormone and calcitonin with regard to cAMP production and⁴⁵Ca release from devitalized bone is examined and indicates that cells from Peak I and Peak III both respond to parathyroid hormone but only the cells from Peak I respond to calcitonin.     

Changes in type of collagen synthesized by chick fibroblasts in vitro in the presence of 5-bromodeoxyuridine.

Chick embryo fibroblasts cease to synthesize their normal collagen product when grown in the presence of the thymidine analogue, 5-bromodeoxyuridine (BrdU). The drug causes an alteration of synthesis from the normal Type I collagen (alpha1 (I)2alpha2) to a mixture of Type I and Type I trimer (alpha1(I)3). While the significance of the synthesis of Type I trimer is unclear, it has been noted that chondrocytes synthesize this collagen type following in vitro senescence and in the presence of BrdU. Since BrdU may cause a switching in the temporal pattern of collagen biosynthesis in chondrocytes and in fibroblasts it is proposed that BrdU may alter the normal regulatory controls acquired by the cells during the course of their differentiation. The synthesis of type I trimer might provide a marker for such a break-down in a wide variety of cell types.     

Production of immunoreactive calcitonin and parathyroid hormone by embryonal carcinoma cells: alteration with retinoic acid-induced differentiation

To determine possible ectopic production of, and altered responsiveness to, specific hormones and growth factors which may be involved in mediating embryonic differentiation and development embryonal carcinoma cells in culture have been employed to serve as an in vitro system of embryogenesis. Exposure of F9 embryonal carcinoma cells to all-trans-retinoic acid previously has been shown to induce differentiation of these undifferentiated stem cells to parietal endoderm and to markedly alter the ability of calcitonin and parathyroid hormone to stimulate adenylate cyclase activity. Evidence is presented that F9 cells secrete immunoreactive calcitonin into the culture medium (200 pg/12 hr/10(7) cells) while parietal yolk sac (PYS) cells secrete immunoreactive parathyroid hormone (800 pg/12 hr/10(7) cells). Retinoic-induced differentiation of F9 cells to endoderm results in a progressive reduction in immunoreactive calcitonin production, while there is an increase in the level of immunoreactive parathyroid hormone found in the conditioned medium. After exposure of F9 cells to retinoic acid for 5 days, little calcitonin is detectable in 12-hr conditioned medium. Changes in the intracellular levels of immunoreactive calcitonin and PTH follow a pattern similar to that noted for changes in the amount of secreted hormones. Thus, immunoreactive calcitonin is produced by undifferentiated F9 cells which possess a calcitonin responsive adenylate cyclase system, while parathyroid hormone is produced by parietal endoderm cells which respond to parathyroid hormone with increased cyclic AMP synthesis. Sephadex G50 gel filtration of F9-conditioned medium shows two peaks of immunoreactive calcitonin with Mr of 3500 and 20,000. Immunoprecipitation of calcitonin from 35S-labeled F9 cells reveals a specific band of 20,000 Mr. Likewise, two peaks of parathyroid hormone immunoreactive material of Mr 8000 and 39,000 are noted after gel filtration of PYS cell-conditioned medium, whereas parathyroid hormone immunoprecipitation from the same cells reveals a specific band of 39,000 Mr. These results raise the possibility that embryo production of these two hormones at specific stages in development may contribute to the regulation of subsequent steps of differentiation.     

The unique action for bone metabolism of 1 alpha,25-(OH)2D3-26,23-lactone

[23 (S), 25 (R)]-1 alpha,25-Dihydroxyvitamin D3-26,23-lactone [( 23 (S),25 (R)]-1 alpha,25-(OH) 2D3-26,23-lactone) increased dose-dependently alkaline phosphatase activity in osteoblastic cells, clone MC3T3-E1, in medium containing 0.1% bovine serum albumin. The maximal stimulated enzyme activity per mg protein was 1.6-fold over that of control cultures at 250 pg/ml. The metabolite also increased collagen synthesis in a dose-related fashion. On the other hand, [23 (S),25 (R)]-1 alpha,25-(OH)2D3-26,23-lactone decreased slightly but significantly 45Ca mobilization, and blocked the resorptive action of 1 alpha,25-dihydroxyvitamin D3 but not that of parathyroid hormone, in mouse calvaria in organ culture. These results indicate that [23 (S),25 (R)]-1 alpha, 25-(OH)2D3-26,23-lactone stimulates the differentiation of osteoblasts and inhibits bone resorption in vitro.     

Regulation of insulin secretion by energy metabolism in pancreatic B-cell mitochondria. Studies with a non-metabolizable leucine analogue.

This study compares the collagen types present in rabbit ear cartilage with those synthesized by dissociated chondrocytes in cell culture. The cartilage was first extracted with 4M-guanidinium chloride to remove proteoglycans. This step also extracted type I collagen. After pepsin solubilization of the residue, three additional, genetically distinct collagen types could be separated by fractional salt precipitation. On SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis they were identified as type II collagen, (1 alpha, 2 alpha, 3 alpha) collagen and M-collagen fragments, a collagen pattern identical with that found in hyaline cartilage. Types I, II, (1 alpha, 2 alpha, 3 alpha) and M-collagen fragments represent 20, 75, 3.5, and 1% respectively of the total collagen. In frozen sections of ear cartilage, type II collagen was located by immunofluorescence staining in the extracellular matrix, whereas type I collagen was closely associated with the chondrocytes. Within 24h after release from elastic cartilage by enzymic digestion, auricular chondrocytes began to synthesize type III collagen, in addition to the above-mentioned collagens. This was shown after labelling of freshly dissociated chondrocytes with [3H]proline 1 day after plating, fractionation of the pepsin-treated collagens from medium and cell layer by NaCl precipitation, and analysis of the fractions by CM(carboxymethyl)-cellulose chromatography and SDS/polyacrylamide-gel electrophoresis. The 0.8 M-NaCl precipitate of cell-layer extracts consisted predominantly of type II collagen. The 0.8 M-NaCl precipitate obtained from the medium contained type I, II, and III collagen. In the supernatant of the 0.8 M-NaCl precipitation remained, both in the cell extract and medium, predominantly 1 alpha-, 2 alpha-, and 3 alpha-chains and M-collagen fragments. These results indicate that auricular chondrocytes are similar to chondrocytes from hyaline cartilage in that they produce, with the exception of type I collagen, the same collagen types in vivo, but change their cellular phenotype more rapidly after transfer to monolayer culture, as indicated by the prompt onset of type III collagen synthesis.     

Cartilage-specific 5' end of chick alpha 2(I) collagen mRNAs

Chondrocytes grown in suspension contain both type I and type II collagen mRNAs, yet synthesize only type II collagen. The inability of chondrocytes to synthesize the alpha 2 subunit of type I collagen, alpha 2(I), results from a severely reduced translation elongation rate (Bennett, V.D., and Adams, S.L. (1987) J. Biol. Chem. 262, 14806-14814). Furthermore, the alpha 2(I) collagen mRNAs from chondrocytes are translated inefficiently in vitro and appear slightly smaller than those from other cells (Focht, R.J., and Adams, S.L. (1984) Mol. Cell. Biol. 4, 1843-1852). These observations suggest that the reduced translation elongation rate may be due to an intrinsic property of the mRNAs. In this report we demonstrate that the alpha 2(I) collagen mRNAs from suspended chondrocytes are 120 bases shorter than those from other cells, and that the first 94 bases of the chondrocyte mRNAs differ from the corresponding region of the calvaria mRNAs. The unique 5' end of the chondrocyte alpha 2(I) collagen mRNAs accounts for their smaller size and may be responsible for the translation elongation defect. Interestingly, the alpha 2(I) collagen mRNAs from chondrocytes grown in monolayer, rather than in suspension, no longer display the cartilage-specific 5' end, suggesting that cell shape and/or adhesion may modulate the structure of the 5' end of the chondrocyte alpha 2(I) collagen mRNAs.     

Regulation of collagen production and collagen mRNA amounts in fibroblasts in response to culture conditions.

Collagen synthesis and mRNA amounts for the alpha 1 and alpha 2 polypeptide chains of Type I collagen were measured in embryonic-chick tendons and in tendon cells both in suspension and in primary cultures. The percentage of protein production represented by collagen in suspension-cultured cells was initially the same as in the intact tendon; however, on an hourly basis, there was actually a steady decline in collagen production by suspended cells. Collagen production in primary cultures of chick tendon fibroblasts was decreased when compared with intact tendon, even though ascorbate-supplemented primary cultures were able to maintain higher rates of collagen production than were non-supplemented cultures. The amounts of mRNA for alpha 1(I) and alpha 2(I) polypeptide chains of collagen responded in similar fashions to different culture conditions and were compared with the amounts of mRNA for beta-actin. In primary cultures the available alpha 1 and alpha 2 collagen mRNAs support proportionately higher collagen production than in the intact tendon. However, the ratio of alpha 1/alpha 2 mRNA and polypeptide-chain synthesis did not remain 2:1, but increased with the concomitant production of Type I trimers composed of three alpha 1 chains. Removal of fibroblasts from their environment in vivo appears to alter the amounts of mRNA for alpha 1 and alpha 2 chains and to alter the utilization of those mRNAs for polypeptide synthesis.     

Change in the activity of calcium ions during illumination of a suspension of visual cell outer segment fragments]

Changes in the activity of calcium ions in the medium containing outer fragments suspension of bovine eye retina rods have been studied by the method of calcium-selective electrodes. Illumination of the suspension increases calcium ion activity in the incubation medium. Photoinduced yield of calcium ions depends on Ca+2 concentration: it equals 0.11+/-0.015 M Ca2+/1m rodopsin in the medium containing 0.1 mM CaCl2 and 0.046+/-0.002Ca2+/1M rodopsin in the medium containing 0.05 mM CaCl2. In the medium containing more than 10(-4) M CaCl2 both an increase and a decrease of Ca2+ ions have been observed.     

Collagen synthesis by human amniotic fluid cells in culture: characterization of a procollagen with three identical proalpha1(I) chains.

Second trimester human amniotic fluid cells synthesize and secrete a variety of collagenous proteins in culture. F cells (amniotic fluid fibroblasts) are the most active biosynthetically and synthesize predominantly type I with smaller amounts of type III procollagen. Epithelioid AF cells (the predominating clonable cell type) synthesize a type IV-like procollagen and a procollagen with three identical proalpha chains, structurally and immunologically related to the proalpha1 chains of type I procollagen. The latter procollagen, when cleaved with pepsin and denatured, yields a single non-disulfide-bonded alpha chain that migrates more slowly than F cell or human skin alpha1(I) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis but coelutes with these chains from carboxymethyl-cellulose. The major cyanogen bromide produced peptides demonstrate a similar behavior relative to peptides derived from alpha1(I). The collagen is characterized by an increased solubility at neutral pH and high ionic strength, relative to type I collagen. The amino acid composition of the pepsin-resistant alpha chain is essentially identical with that of human alpha1(I), except for marked increases in the content of 3- and 4-hydroxyproline and hydroxylysine. Preliminary experiments suggest that these increased posttranslational modifications are responsible for the unusually slow migration of this collagen and its cyanogen bromide peptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The procollagen has, therefore, been assigned the chain composition [proalpha1(I)]3. Like type I procollagen, [proalpha1(I)]3 undergoes a time-dependent conversion, in the medium and cell layer, to procollagen intermediates and alpha chains. The production of [proalpha1(I)]3 probably reflects the state of differentiation and/or embryologic derivation of AF cells rather than a characteristic of the fetal phenotype, since F cells do not synthesize significant amounts of the procollagen.     

Improved maintenance of adult rat alveolar type II cell differentiation in vitro: effect of hydrocortisone and cyclic AMP

We have examined the effect of hydrocortisone and cyclic AMP on the maintenance of lipid synthesis in primary cultures of adult rat alveolar type II cells. These hormones were tested in the presence of either 1% or 5% charcoal-stripped rat serum (CS-rat serum). The effect of substratum on responsiveness to these hormones was evaluated by comparing cells cultured for 4 days on tissue culture plastic, on floating type I collagen gels, on rat lung fibroblast feeder layers on floating collagen gels (floating feeder layers), and on Engelbreth-Holm-Swarm (EHS) tumor basement membrane gels. Type II cells cultured on floating feeder layers in medium containing 1% CS-rat serum and 10(-5) M hydrocortisone plus 0.5 mM dibutyryl cyclic AMP exhibited significantly increased incorporation of [14C]acetate into total lipids (238% of control). The hormone combination also increased the relative percentage of acetate incorporated into phosphatidylglycerol (PG; 7.3% versus 1.9%) and saturated phosphatidylcholine (PC; 43.6% versus 37.6%). The percentage of acetate incorporated into neutral lipids was significantly decreased by the addition of hormones (28.6% versus 70.0%). The addition of hydrocortisone and cyclic AMP to medium containing 5% CS-rat serum resulted in an increase in the relative incorporation of acetate into saturated PC (51.2% versus 46.4%), but had no effect on the relative incorporation of acetate into PG or on the incorporation of acetate into total lipids. Type II cells cultured on EHS gels in medium containing 1% CS-rat serum plus hydrocortisone and cyclic AMP showed increased acetate incorporation into total lipids (204% of control) and a relative decrease in the percentage of acetate incorporated into neutral lipids (16.9% versus 47.0%). The hormone combination also increased the relative incorporation of acetate into PG (4.4% versus 2.5%) and saturated PC (49.9% versus 42.1%). Hydrocortisone and cyclic AMP added to medium containing 5% CS-rat serum concentration increased the relative incorporation of acetate into saturated PC by type II cells on EHS gels, but these additions had no effect on acetate incorporation into PG. No responses to these soluble factors were seen when type II cells were cultured on floating type I collagen gels without feeder layers or on tissue culture plastic. These data indicate that there are positive interactions between substratum, soluble factors and serum in the maintenance of differentiated function of adult rat alveolar type II cells in vitro.     

Effect of 4-methylumbelliferyl-β-d-xyloside on collagen synthesis in chick limb bud mesenchymal cell cultures

Previous studies showed that cultures of chick limb bud mesenchymal cells plated at high density, to maximize chondrogenic expression, had a much reduced extracellular matrix around chondrocytes when exposed to 4-methyl-, umbelliferyl-β-d-xyloside. The majority of newly synthesized chondroitin sulfate chains were found in the culture medium presumably bound to the xyloside as opposed to their normal deposition on the core protein of proteoglycan. The question remained open as to whether the development of an abnormal matrix affected the synthesis of extracellular deposition of other cartilage-specific macromolecules. We have analyzed, both morphologically and biochemically, the synthesis and deposition of Type I and Type II collagen by β-d-xyloside-treated cultures of limb mesenchymal cells. While the rate of collagen synthesis per plate and its extracellular accumulation after 8 days in culture were reduced to some extent, the ratios of Type II to Type I collagen and the morphological distribution of these macromolecules were not affected by exposure to β-d-xyloside. We conclude that the expression of the cartilage-specific Type II collagen during chondrogenic differentiation is, although reduced, qualitatively not dependent on the amount of extracellular chondroitin sulfate chains attached to matrix-associated proteoglycan core protein. However, prolonged exposure of limb bud cells to xylosides leads to the formation of a chondroitin sulfate- and collagen-deficient matrix which, in turn, reduces the capacity of limb bud cells to synthesize Types I and II collagen.     

RGDS and DGEA-induced [Ca2+]i signalling in human dermal fibroblasts

A pulse of short peptides, RGDS and DGEA in the millimolar range, immediately elicits in normal human fibroblasts a transient increase of intracellular Ca2+ ([Ca2+]i). In the present study, we show that this [Ca2+]i occurs in an increasing number of cells as a function of peptides concentration. It is specific of each peptide and inhibited at saturating concentration of the peptide in the culture medium. The [Ca2+]i transient depends on signalling pathways slightly different for DGEA and RGDS involving tyrosine kinase(s) and phosphatase(s), phospholipase C, production of inositol-trisphosphate and release of Ca2+ from the cellular stores. GFOGER, the classical collagen binding peptide of alpha1- alpha2- and alpha11-beta1 integrins, in triple helical or denatured form, does not produce any Ca2+ signal. The [Ca2+]i signalling induced by RGDS and DGEA is inhibited by antibodies against beta1 integrin subunit while that mediated by RGDS is also inhibited by antibodies against the alpha3 integrin. Delay in the acquisition of responsiveness is observed during cell adhesion and spreading on a coat of fibronectin for RGDS or collagen for DGEA or on a coat of the specific integrin-inhibiting antibodies but not by seeding cells on GFOGER or laminin-5. This delay is suppressed specifically by collagenase acting on the collagen coat or trypsin on the fibronectin coat. Our results suggest that free integrins and associated focal complexes generate a Ca2+ signal upon recognition of DGEA and RGDS by different cellular pathways.