Growth hormone-releasing peptide-2 stimulates secretion and synthesis of adrenocorticotropic hormone in mouse pituitary

Growth hormone (GH)-releasing peptides (GHRPs) are synthetic peptides which induce strong GH release in both animals and humans. Among them, GHRP-2 is known to stimulate GH release by acting at both hypothalamic and pituitary sites, but also induces adrenocorticotropic hormone (ACTH) release in healthy subjects. GHRP-2 may stimulate ACTH release directly via GHRP receptor type 1a in ACTH-producing tumors. GHRP-2 increases ACTH secretion in rat in vivo, but not ACTH release from rat primary pituitary cells. In the present study, in order to elucidate the mechanism underlying ACTH secretion by GHRPs, mouse pituitary cells were stimulated by GHRP-2. GHRP receptor mRNA was expressed in the mouse pituitary, and GHRP-2 directly stimulated secretion and synthesis of ACTH in the mouse anterior pituitary cells. GHRP-2 increased intracellular cyclic AMP production. H89, a potent protein kinase A (PKA) inhibitor, and bisindolylmaleimide I, a selective protein kinase C (PKC) inhibitor, inhibited the GHRP-2-induced ACTH release, and that H89, but not bisindolylmaleimide I, inhibited the GHRP-2-induced proopiomelanocortin mRNA levels. Together, the GHRP-2-induced ACTH release was regulated via both PKA and PKC pathways in the mouse pituitary cells, while ACTH was synthesized by GHRP-2 only via the PKA pathway.     

Extracellular matrix components direct porcine muscle stem cell behavior

In muscle tissue, extracellular matrix proteins, together with the vasculature system, muscle-residence cells and muscle fibers, create the niche for muscle stem cells. The niche is important in controlling proliferation and directing differentiation of muscle stem cells to sustain muscle tissue. Mimicking the extracellular muscle environment improves tools exploring the behavior of primary muscle cells. Optimizing cell culture conditions to maintain muscle commitment is important in stem cell-based studies concerning toxicology screening, ex vivo skeletal muscle tissue engineering and in the enhancement of clinical efficiency. We used the muscle extracellular matrix proteins collagen type I, fibronectin, laminin, and also gelatin and Matrigel as surface coatings of tissue culture plastic to resemble the muscle extracellular matrix. Several important factors that determine myogenic commitment of the primary muscle cells were characterized by quantitative real-time RT-PCR and immunofluorescence. Adhesion of high PAX7 expressing satellite cells was improved if the cells were cultured on fibronectin or laminin coatings. Cells cultured on Matrigel and laminin coatings showed dominant integrin expression levels and exhibited an activated Wnt pathway. Under these conditions both stem cell proliferation and myogenic differentiation capacity were superior if compared to cells cultured on collagen type I, fibronectin and gelatin. In conclusion, Matrigel and laminin are the preferred coatings to sustain the proliferation and myogenic differentiation capacity of the primary porcine muscle stem cells, when cells are removed from their natural environment for in vitro culture.     

Cell adhesion to collagen and decreased myogenic gene expression implicated in the control of myogenesis by transforming growth factor beta

Transforming growth factor beta 1 (TGF-beta 1) is an inhibitor of skeletal muscle myoblast differentiation. Myoblast differentiation is dependent on the expression of certain myogenic differentiation genes and is affected by cell interaction with the extracellular matrix. We have searched for events in the differentiation process of L6E9 rat myoblasts that may be involved in the inhibitory action of TGF-beta 1. Elevated expression of the myogenic differentiation gene, myogenin, which is thought to play a central role in the differentiation process, occurs 10 h after the shift of L6E9 myoblasts to differentiation medium. Elevation of myogenin mRNA is blocked by TGF-beta 1 added at the time of the shift. This effect is preceded by, and might be related to, a rapid up-regulation of junB mRNA observed in TGF-beta 1-treated L6E9 myoblasts. However, TGF-beta 1 can also block myogenic differentiation in cells transfected with the myogenin gene under the control of a constitutive SV40 viral promoter. The nature of a mechanism that could mediate the inhibitory action of TGF-beta 1 without blocking myogenin mRNA expression is suggested by the observations that (a) TGF-beta 1 upregulates type I collagen expression and deposition in L6E9 myoblasts, (b) a fibrillar type I collagen layer inhibits L6E9 myoblast differentiation, and (c) inhibition of L6E9 myoblast differentiation by a type I collagen layer occurs without a block in myogenin expression. Thus, the data suggest that inhibition of L6E9 myoblast differentiation by TGF-beta 1 may be accomplished by at least two mechanisms acting in concert. One mechanism leads to a block in the expression of myogenin whereas the other mechanism may involve TGF-beta 1-induced changes in cell adhesion that either block the action of myogenic differentiation gene products or prevent the function of other as yet unknown components of the myogenic differentiation pathway.     

Colonization of the satellite cell niche by skeletal muscle progenitor cells depends on notch signals

Skeletal muscle growth and regeneration rely on myogenic progenitor and satellite cells, the stem cells of postnatal muscle. Elimination of Notch signals during mouse development results in premature differentiation of myogenic progenitors and formation of very small muscle groups. Here we show that this drastic effect is rescued by mutation of the muscle differentiation factor MyoD. However, rescued myogenic progenitors do not assume?a satellite cell position and contribute poorly to myofiber growth. The disrupted homing is due to a deficit in basal lamina assembly around emerging satellite cells and to their impaired adhesion to myofibers. On a molecular level, emerging satellite cells deregulate the expression of basal lamina components and adhesion molecules like integrin α7, collagen XVIIIα1, Megf10, and Mcam. We conclude that Notch signals control homing of satellite cells, stimulating them to?contribute to their own microenvironment and to adhere to myofibers.     

Synthesis of type IV collagen and laminin in cultures of skeletal muscle cells and their assembly on the surface of myotubes

The synthesis of two components of the basal lamina, laminin and type IV collagen, and their extracellular deposition on the surface of myotubes was studied in cultures of embryonic mouse and quail skeletal muscle cells and in the rat myoblast cell line L6. Production of type IV collagen and laminin by myoblasts and muscle fibroblasts was demonstrated by incorporation of radioactive amino acids into proteins and by immunoprecipitation with specific antibodies and electrophoretic analysis of labeled proteins. Immunofluorescence staining experiments revealed strong intracellular reactions with antibodies to laminin and type IV collagen in mononucleated myogenic and fibrogenic cells. Cells of fibroblast-like morphology showed a more intense staining than bipolar, spindle-shaped cells which perhaps represented postmitotic myoblasts. Myotubes did not show detectable intracellular staining. The formation of a basal lamina on myotubes was indicated by the deposition of laminin and type IV collagen on the surface of myotubes as viewed by immunofluorescence examination of unfixed cells. Staining for extracellular laminin was stronger in mass cultures than in myogenic clones, suggesting that secretion and deposition of components of the basal lamina on the myotube surface are complex processes which may involve cooperation between myogenic and fibrogenic cells.     

Growth on type I collagen promotes expression of the osteoblastic phenotype in human osteosarcoma MG-63 cells.

Using MG-63 cells as a model system capable of partial osteoblastic differentiation, we have examined the effect of growth on extracellular matrix. MG-63 cell matrix and purified type I collagen induced a morphological change characterized by long cytoplasmic processes reminiscent of those seen in osteocytes. Concurrent biochemical changes involving bone marker proteins included increased specific activity of cell-associated alkaline phosphatase and increased secretion of osteonectin (up to 2.5-fold for each protein); all changes occurred without alterations in the growth kinetics of the MG-63 cells. The increase in alkaline phosphatase activity was maximal on days 6-8 following seeding; increased osteonectin secretion was most prominent immediately following seeding; all changes decreased as cells reached confluence. Growing cells on type I collagen resulted in an increased induction of alkaline phosphatase activity by 1,25(OH)2D3 (with little change in the 1,25(OH)2D3 induction of osteonectin and osteocalcin secretion), and increased TGF-beta induction of alkaline phosphatase activity as well (both TGF-beta 1 and TGF-beta 2). Both the 1,25(OH)2D3 and TGF-beta effects appeared to be synergistic with growth on type I collagen. These studies support the hypothesis that bone extracellular matrix may play an important role in osteoblastic differentiation and phenotypic expression.     

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.     

Implication of the satellite cell in dystrophic muscle fibrosis: a self-perpetuating mechanism of collagen overproduction

Because of its mechanical function, skeletal muscle is heavily influenced by the composition of its extracellular matrix (ECM). Fibrosis generated by chronic damage, such as occurs in muscular dystrophies, is thus particularly disastrous in this tissue. Here, we examined the interrelationship between the muscle satellite cell and the production of collagen type I, a major component of fibrotic ECM, by using both C2C12, a satellite cell-derived cell line, and primary muscle satellite cells. In C2C12 cells, we found that expression of collagen type I mRNA decreases substantially during skeletal muscle differentiation. On a single-cell level, collagen type I and myogenin became mutually exclusive after 3 days in differentiation medium, whereas addition of collagen markedly suppressed differentiation of C2C12 cells. Primary cultures of satellite cells associated with isolated single fibers of the young (4 wk old) mdx dystrophic mouse and of C57BL/10ScSn wild-type controls expressed collagen type I and type III mRNA and protein. This pattern persisted in wild-type mice at all ages. But, curiously, in older (18-mo-old) mdx mice, although the myogenic cells continued to express type III collagen, type I expression became restricted to nonmyogenic cells. These cells typically constituted part of a cellular sheet surrounding the old mdx fibers. This combination of features strongly suggests that the progression to fibrosis in dystrophic muscle involves changes in the mechanisms controlling matrix production, which generates positive feedback that results in a reprogramming of myoblasts to a profibrotic function. collagen type I; myogenin; muscle single fibers; Duchenne muscular dystrophy     

Laminin alpha4 and integrin alpha6 are upregulated in regenerating dy/dy skeletal muscle: comparative expression of laminin and integrin isoforms in muscles regenerating after crush injury

The expression of laminin isoforms and laminin-binding integrin receptors known to occur in muscle was investigated during myogenic regeneration after crush injury. Comparisons were made between dystrophic 129ReJ dy/dy mice, which have reduced laminin alpha2 expression, and their normal littermates. The overall histological pattern of regeneration after crush injury was similar in dy/dy and control muscle, but proceeded faster in dy/dy mice. In vitro studies revealed a greater yield of mononuclear cells extracted from dy/dy muscle and a reduced proportion of desmin-positive cells upon in vitro cultivation, reflecting the presence of inflammatory cells and "preactivated" myoblasts due to ongoing regenerative processes within the endogenous dystrophic lesions. Laminin alpha1 was not detectable in skeletal muscle. Laminin alpha2 was present in basement membranes of mature myofibers and newly formed myotubes in control and dy/dy muscles, albeit weaker in dy/dy. Laminin alpha2-negative myogenic cells were detected in dy/dy and control muscle, suggesting the involvement of other laminin alpha chains in early myogenic differentiation, such as laminin alpha4 and alpha5 which were both transiently expressed in basement membranes of newly formed myotubes of dy/dy and control mice. Integrin beta1 was expressed on endothelial cells, muscle fibers, and peripheral nerves in uninjured muscle and broadened after crush injury to the interstitium where it occurred on myogenic and nonmyogenic cells. Integrin alpha3 was not expressed in uninjured or regenerating muscle, while integrin alpha6 was expressed mainly on endothelial cells and peripheral nerves in uninjured muscle. Upon crush injury integrin alpha6 increased in the interstitium mainly on nonmyogenic cells, including infiltrating leukocytes, endothelial cells, and fibroblasts. In dy/dy muscle, integrin alpha6 occurred on some newly formed myotubes. Integrin alpha7 was expressed on muscle fibers at the myotendinous junction and showed weak and irregular expression on muscle fibers. After crush injury, integrin alpha7 expression extended to the newly formed myotubes and some myoblasts. However, many myoblasts and newly formed myotubes were integrin alpha7 negative. No marked difference was observed in integrin alpha7 expression between dy/dy and control muscle, either uninjured or after crush injury. Only laminin alpha4 and integrin alpha6 expression patterns were notably different between dy/dy and control muscle. Expression of both molecules was more extensive in dy/dy muscle, especially in the interstitium of regenerating areas and on newly formed myotubes. In view of the faster myogenic regeneration observed in dy/dy mice, the data suggest that laminin alpha4 and integrin alpha6 support myogenic regeneration. However, whether these accelerated myogenic effects are a direct consequence of the reduced laminin alpha2 expression in dy/dy mice, or an accentuation of the ongoing regenerative events in focal lesions in the muscle, requires further investigation.     

Developmental regulation of a secreted gelatin-binding protein during myogenesis in vitro

Collagen has a stimulatory effect on the differentiation of skeletal muscle cells in culture. Putative collagen-binding proteins were isolated from detergent-solubilized cultures of the L6 rat muscle cell line and primary clonal cultures of human skeletal muscle satellite cells, using gelatin-Sepharose affinity chromatography. In addition to fibronectin, which has been reported by others to be synthesized by cultured muscle cells, we found that muscle cultures synthesized gelatin-binding proteins of lower apparent molecular weight. Only one of these proteins was secreted into the growth medium and bound to type I collagen. Binding of this protein to gelatin and collagen-Sepharose was resistant to repeated washing with 1 M NaCl and nonionic detergent. The secreted gelatin-binding protein had an apparent molecular weight of 63,000-72,000, depending upon the conditions of electrophoresis. The lack of reactivity of the secreted protein with polyclonal antisera against fibronectin, the lack of effect of protease inhibitors on its appearance in the medium, and the rapid de novo production of the protein during pulse labeling with radioactive methionine indicated that it was not a fibronectin fragment. The rate of synthesis of the secreted gelatin-binding protein increased markedly during the myogenesis of rat and human cultures.     

Myotube morphology, and expression and distribution of collagen type I during normal and low score normal avian satellite cell myogenesis

Myogenic satellite cells are essential for postnatal muscle growth and the regeneration of muscle in response to injury. An understanding of how the extracellular matrix affects satellite cell activity, and the temporal and spatial expression of extracellular matrix macromolecules is largely unknown. In the avian genetic muscle weakness, low score normal (LSN), satellite cell proliferation and differentiation rates are significantly lower than that observed in normal chicken satellite cells, which may be attributed to a late embryonic increase in the expression of decorin. Satellite cell-derived morphological properties, collagen type I expression, and the spatial distribution of collagen type I were investigated during normal and LSN satellite cell proliferation and differentiation. These studies showed a decrease in LSN myotube length and the number of nuclei per myotube. Collagen type I expression was similar between the LSN and normal satellite cell cultures during the course of proliferation and differentiation. However, the spatial distribution of collagen type I was altered in the LSN cultures 48 h after the initiation of fusion. The LSN cultures exhibited a premature extracellular distribution of collagen type I compared to the normal satellite cells.     

Endoglin differentially modulates antagonistic transforming growth factor-beta1 and BMP-7 signaling

Transforming growth factor-beta1 (TGF-beta1) and BMP-7 (bone morphogenetic protein-7; OP-1) play central, antagonistic roles in kidney fibrosis, a setting in which the expression of endoglin (CD105), an accessory TGF-beta type III receptor, is increased. So far, endoglin is known as a negative regulator of TGF-beta/ALK-5 signaling. Here we analyzed the effect of BMP-7 on TGF-beta1 signaling and the role of endoglin for both pathways in endoglin-deficient L(6)E(9) cells. In this myoblastic cell line, TGF-beta1 and BMPs are opposing cytokines, interfering with myogenic differentiation. Both induce specific target genes of which Id1 (for BMPs) and collagen I (for TGF-beta1) are two examples. TGF-beta1 activated two distinct type I receptors, ALK-5 and ALK-1, in these cells. Although the ALK-5/Smad3 signaling pathway mediated collagen I expression, ALK-1/Smad1/Smad5 signaling mediated a transient Id1 up-regulation. In contrast, BMP-7 exclusively activated Smad1/Smad5 resulting in a more prolonged Id1 expression. Although BMP-7 had no impact on collagen I abundance, it antagonized TGF-beta1-induced collagen I expression and (CAGA)(12)-MLP-Luc activity, effects that are mediated by the ALK-5/Smad3 pathway. Finally, we found that the transient overexpression of endoglin, previously shown to inhibit TGF-beta1-induced ALK-5/Smad3 signaling, enhanced the BMP-7/Smad1/Smad5 pathway.     

Recruitment of Type I Collagen Producing Cells from the Microvasculaturein Vitro

We have previously suggested that microvascular pericytes can differentiate into fibroblast-like, type I collagen-producing cells during excessive dermal scarringin vivo(Sundberg, C., Ivarsson, M., Gerdin, B., and Rubin, K.,Lab. Invest.74, 454–468, 1996). Here we have investigated to what extent pericytes derived from microvessels of full-term human placenta exhibited this capacityin vitro.Vascular fragments of human term placenta were isolated by enzymatic digestion and separation in Percoll. Their microvascular origin was ascertained by confocal microscopy using antibodies specific for endothelial cells (PAL-E) and pericytes (high-molecular-weight–melanoma-associated antigen). When vascular fragments were culturedin vitro,large cells with irregular edges migrated out from the fragments. After 4–6 days in culture, these cells started to proliferate and reached near confluence after approximately 8 days. The cultures were not overgrown by clones of cells with a high proliferative capacity, as demonstrated by cell membrane fluorescence staining and Ki67 expression. Expression of PAL-E, high-molecular-weight–melanoma-associated antigen, smooth muscle α-actin, desmin, and collagen synthesis (prolyl-4-hydroxylase and type I procollagen, as well as collagen pro-α1(I) mRNA) were followed during a culture period of 8 days. The cells were PAL-E negative but expressed high-molecular-weight–melanoma-associated antigen, smooth muscle α-actin, and desmin. Based on morphology and expression of the various markers, the outgrowing cells were identified as pericytes. With time in culture the cells decreased their expression of all these markers and increased their expression of prolyl-4-hydroxylase, type I procollagen, and collagen pro-α1(I) mRNA. Metabolic labeling and SDS–PAGE analysis of labeled proteins revealed that type I collagen was the major collagen species synthesized in the cultures. Our results support the hypotheses that pericytes can leave the vasculature and differentiate into collagen-producing cells and that cultured “fibroblasts” are derived from pericytes.     

Effect of relaxin on the phenotype of collagens synthesized by cultured rabbit chondrocytes

The effect of porcine relaxin on rabbit articular and growth plate chondrocytes in primary culture was investigated by measurement of total collagen production and analysis of the phenotypes of newly synthesized collagen chains. A 24-h treatment of monolayer articular and multilayer growth plate chondrocytes with 2 micrograms per ml relaxin had no effect on total DNA and did not significantly modify the amount of [3H]proline-labelled collagen chains secreted by the cells. However, polyacrylamide gel electrophoresis demonstrated relevant modifications in relaxin treated chondrocytes. A significant increase was observed in the proportion of type III collagen and in the intensity of the band corresponding to alpha 2I chains. Two-dimensional peptide mapping of CNBr-cleaved molecules indicated that the band that was identified as alpha 1II on monodimensional gels contained a significant proportion of alpha 1I collagen chains, as demonstrated by the presence of alpha 1I cyanogen bromide-digested peptides. The intensity of this band was increased by relaxin treatment. Furthermore, total RNA analysis by slot blot and Northern blot techniques showed a dose-dependent stimulation of alpha 1I and alpha 1III mRNA levels after incubation with increased relaxin concentrations, but no change in the amount of alpha 1II mRNA. These results suggested that when added to cartilage cells in vitro, relaxin modulated the expression of type I, type II and type III collagen genes by amplifying the dedifferentiation process.     

Mouse teratocarcinoma and embryonic development. Two-dimensional protein patterns in muscle differentiation

Muscle cell differentiation was analysed by two-dimensional gel electrophoresis of newly synthesized proteins compared in two parallel systems: in mouse teratocarcinoma-derived tumors that became restricted in their developmental potential to the formation of muscle-like cells and in developing limbs of the early mouse fetus. Muscle cell differentiation in teratocarcinomas was found to be reflected by both a marked decrease in the rate of synthesis of about 12% (119 proteins) of the resolved polypeptides and a pronounced increase in the synthesis of another large set of proteins (83 proteins). The majority of the newly acquired proteins (46 proteins) were also detected in fetal brain and muscle tissue. These proteins are considered to be those which accompany differentiation in general, regardless of cell type, as e.g. ubiquitously occurring structural proteins. Their expression in the muscle cells of the tumors may reflect the normal aspect of this particular differentiation pathway. Five polypeptides were found to appear specifically in both myogenic tumors and developing limbs, but not during brain formation and were tentatively termed muscle-specific proteins (MSP). The identification of differentiation-related proteins in muscle development will allow us to analyse differentiation in early development in molecular terms.     

Ghrelin and its unacylated isoform stimulate the growth of adrenocortical tumor cells via an anti-apoptotic pathway

Ghrelin is expressed in normal human adrenocortical cells and induces their proliferation through growth hormone secretagogue receptor 1a (GHS-R1a). Consequently, it was of interest to us to determine whether acylated ghrelin and its predominant serum isoform, unacylated ghrelin, also act as factors for adrenocortical carcinoma cell growth. To examine a potential ghrelin-regulated system in adrenocortical tumors, we measured proliferative effects of acylated and unacylated ghrelin in the adrenocortical carcinoma cell lines SW-13 and NCI-H295R. We also examined the expression of ghrelin, GHS-R1a, and corticotrophin-releasing factor receptor 2 (CRF-R2). Acylated and unacylated ghrelin in the nanomolar range dose-dependently induced adrenocortical cell growth up to 200% of untreated controls, as measured by thymidine uptake and WST1 assay. The proliferative effects of acylated and unacylated ghrelin in SW-13 cells was blocked by [D-Lys(3)]growth hormone-releasing peptide 6 (GHRP6), but a CRF-R2 antagonist had no effect on unacylated ghrelin growth stimulation. Cell cycle analysis suggests that acylated and unacylated ghrelin suppress the sub-G(0)/apoptotic fraction by up to 50%. Measurement of DNA fragmentation and caspase-3 and -7 activity in SW-13 cells confirmed that acylated and unacylated ghrelin suppress apoptotic rate. SW-13 cells express preproghrelin mRNA and secrete ghrelin, and [D-Lys(3)]GHRP6 suppresses their basal proliferation rate, strongly suggesting that ghrelin could act as an auto/paracrine growth factor. Acylated and unacylated ghrelin are potential auto/paracrine factors acting through an antiapoptotic pathway to stimulate adrenocortical tumor cell growth. Unacylated ghrelin-stimulated growth is suppressed by an antagonist of GHS-R1a, suggesting either that unacylated ghrelin is acylated before its action or that ghrelin, unacylated ghrelin, and [D-Lys(3)]GHRP-6 bind to a novel receptor in these cells.     

Bleomycin-induced synthesis of type I procollagen by human lung and skin fibroblasts in culture

Bleomycin is a chemotherapeutic agent sometimes associated with pulmonary fibrosis and skin lesions in patients undergoing treatment. We examined the mechanisms of increased collagen deposition on bleomycin-induced fibrosis by incubating human lung and skin fibroblast cultures with [14C]proline; the synthesis of [14C]hydroxyproline relative to DNA or cell protein was taken as an index of procollagen formation. Procollagen synthesis by lung cells in the presence of 0.1 and 1.0 microgram/ml bleomycin was significantly increased and similar results were obtained with skin fibroblasts. The relative synthesis of genetically distinct types of collagen was measured by isolating the newly synthesized type I and type III procollagens by DEAE-cellulose chromatography. The proportion of type III procollagen of total newly synthesized procollagen in control lung fibroblast cultures was 17.4 +/0 0.6% (mean +/- S.E.) while the corresponding value in cells incubated in 1 microgram/ml bleomycin was 12.5 +/- 0.6% (n = 6, P < 0.01). Similar results were obtained when the ratios of newly synthesized type I and type III collagens were estimated by interrupted polyacrylamide disc gel electrophoresis in sodium dodecyl sulfate after a limited proteolytic digestion with pepsin. The results indicate that the increased procollagen synthesis induced by bleomycin in fibroblast cultures is predominantly directed towards the synthesis of type I procollagen.     

Type X collagen expression in osteoarthritic and rheumatoid articular cartilage

Type X collagen is a short chain, non-fibrilforming collagen synthesized primarily by hypertrophic chondrocytes in the growth plate of fetal cartilage. Previously, we have also identified type X collagen in the extracellular matrix of fibrillated, osteoarthritic but not in normal articular cartilage using biochemical and immunohistochemical techniques (von der Mark et al. 1992 a). Here we compare the expression of type X with types I and II collagen in normal and degenerate human articular cartilage by in situ hybridization. Signals for cytoplasmic α1(X) collagen mRNA were not detectable in sections of healthy adult articular cartilage, but few specimens of osteoarthritic articular cartilage showed moderate expression of type X collagen in deep zones, but not in the upper fibrillated zone where type X collagen was detected by immunofluorescence. This apparent discrepancy may be explained by the relatively short phases of type X collagen gene activity in osteoarthritis and the short mRNA half-life compared with the longer half-life of the type X collagen protein. At sites of newly formed osteophytic and repair cartilage, α1(X) mRNA was strongly expressed in hypertrophic cells, marking the areas of endochondral bone formation. As in hypertrophic chondrocytes in the proliferative zone of fetal cartilage, type X collagen expression was also associated with strong type II collagen expression.