Study of subchondral bone adaptations in a rodent surgical model of OA using in vivo micro-computed tomography.

OBJECTIVE: To non-invasively investigate the changes to epiphyseal bone occurring in a longitudinal pre-clinical model of osteoarthritis (OA) using in vivo micro-computed tomography (micro-CT). DESIGN: In vivo micro-CT images were acquired using a bench-top micro-CT scanner, which produces three-dimensional data with isotropic voxel spacing of 0.046 mm. Male rodents were scanned prior to surgical destabilization, consisting of anterior cruciate ligament transection and partial medial menisectomy (ACLX). Subsequent scans were performed every 4 weeks post-ACLX, for up to 5 months. Volumetric bone mineral density (vBMD) was measured in specific, anatomically segmented regions within each image. The ACLX rodent data were compared with the contralateral non-operated hind limb of the same animal, as well as a sham-operated group (SHAM) of animals, for each time point. End-point histology compared changes to cartilage and bone between the ACLX and control animals. RESULTS: The micro-CT protocol produced sufficient spatial resolution and signal-to-noise ratio (SNR=19) to quantify subchondral bone pathology, with an acceptable entrance exposure to radiation (0.36 Gy). Significantly lower vBMD was measured in the ACLX group, vs SHAM rodents, at 1, 4, and 5 months post-surgery (P<0.05). Qualitative observations of ACLX joints revealed significant loss of cartilage, subchondral bone cysts, and calcification of tendon similar to changes found in humans. CONCLUSIONS: This study demonstrates in vivo micro-CT as an effective method for investigating the development of rodent knee OA longitudinally. This method can be applied, in future pre-clinical trials, to non-destructively monitor the efficacy of pharmacological interventions.

Adhesion to fibronectin or collagen I gel induces rapid, extensive, biosynthetic alterations in epithelial cells.

Extracellular matrix influences many cellular events. In this study, we demonstrate that adhesion of human salivary gland (HSG) epithelial cells to fibronectin- or collagen I gel-coated substrates, mediated by beta1 integrins, results in substantial alterations in protein and RNA expression profiles. The large numbers of changes in expression suggest that simply changing the adhesive substrate has basic effects on the regulation of cellular biosynthesis. Two-dimensional electrophoresis of [35S]methionine-labeled HSG cell proteins identified significant differences in the patterns of protein expression by cells cultured on nonprecoated substrates, collagen I gels or fibronectin. Thirty-two differentially expressed cDNA clones, which included both novel and previously sequenced genes, were up-regulated within 6 hr by culturing HSG cells on fibronectin or collagen I gels. Therefore, adhesion to collagen I or fibronectin resulted in rapid, widespread changes in cellular biosynthetic control. Expression of some genes was induced by ligation of beta1 integrins with antifunctional antibodies, whereas the expression of other genes was not induced. Most of the differentially expressed genes were up-regulated by adhesion to both fibronectin- and collagen I gel-coated substrates, but a few genes were selectively up-regulated on only one substrate. Furthermore, the up-regulated expression of some genes was detected within 3 hr, whereas changes in others required 6 hr. Discrete adhesive substrates and integrin molecules differentially affected the expression of a significant number of genes, suggesting that the cellular responses to adhesion were triggered through several signaling pathways.

There is temporal and spatial expression of alpha1 (IV), alpha2 (IV), alpha5 (IV), alpha6 (IV) collagen chains and beta1 integrins during the development of the basal lamina in an "in vitro" skin model.

Temporal and spatial expression of alpha1 (IV), alpha2 (IV), alpha3 (IV), alpha4 (IV), alpha5 (IV), and alpha6 (IV) collagen chains was studied during the formation of the basal lamina in an "in vitro" skin model. A sequential study was performed at 7-d and 14-d cultures (lamina densa absent) and at 28-, 36-, and 56-d cultures (lamina densa present). Expression of beta1, beta4, alpha1, alpha2, alpha3, alpha5, alpha6 integrin subunits and co-localization with collagen IV was studied by regular and laser confocal indirect immunofluorescence microscopy. mRNA expression of alpha2 (IV) and alpha6 (IV) chains was estimated by northern blots. The earliest expression of alpha1 (IV) and alpha2 (IV) collagen chains was noted in 7-d cultures restricted to basal keratinocytes. At 14-d cultures, alpha1 (IV) and alpha2 (IV) chains were noted in basal keratinocytes and as a broad band (10 microm) in the adjacent dermis. At this stage 80% of the alpha2 (IV) mRNA was expressed in the dermis and 20% in the epidermis. At 28-, 36-, and 56-d cultures the alpha1 (IV) and alpha2 (IV) chains were present in a linear distribution at the epidermo-dermal junction and in the upper dermis. The alpha6 (IV) collagen chains were expressed much later at 36-d cultures and the alpha5 (IV) at 56 d, both mostly in a linear distribution but also in the adjacent dermis. Alpha6 (IV) mRNA was demonstrated in the dermis of 36-d cultures. There was co-localization of collagen IV and beta1 integrin subunits in 14-d cultures at the matrix site of keratinocytes. Functional perturbation studies with AIIB2 monoclonal antibody (anti-beta1 subunits) and competitive inhibition with a collagen cyanogen bromide digestion derived fragment (CB3[IV]) that contains the collagen IV ligand for alpha1beta1, alpha2beta1 integrins, altered the pattern of collagen IV deposition.

Identification of a minimum enhancer sequence for the type II collagen gene reveals several core sequence motifs in common with the link protein gene.

The type II collagen gene (Col2a1) is expressed primarily in chondrocytes. Transcription of Col2a1 is mediated by cell-specific regulatory elements located within the promoter and first intron. Here, we map a minimal enhancer and identify elements that determine cartilage-specific Col2a1 expression by analyzing the activity of a series of chimeric genes consisting of rat Col2a1 first intron deletion mutants ligated to the chloramphenicol acetyltransferase reporter gene. We show that a 100-base pair (bp) segment within the first intron is the minimum size necessary for high level, cell type-specific expression of Col2a1. Sequence analysis of this 100-bp Col2a1 enhancer revealed several sequence motifs similar to motifs present within the regulatory region of the link protein gene, another cartilage gene. These motifs include an AT-rich element, a C1 motif and a C3 motif. Deletion of any of these elements reduced Col2a1 enhancer activity in chick embryo chondrocytes. We also tested enhancer-mediated activity in CFK2 cells which differentiate to a chondrogenic phenotype and begin to express type II collagen mRNA after extended culture. In stably transfected CFK2 cells, constructs containing the 100-bp enhancer were activated during the transition from prechondrogenic to chondrogenic cell populations and deletions within the enhancer strongly down-regulated activity. Chondrocyte-specific DNA-protein complexes were identified using nuclear extracts prepared from chick embryo chondrocytes and 32P-labeled oligonucleotides from these regions of the first intron. These results suggest that interaction of chondrocyte specific nuclear factors with multiple core elements from a small region within the first intron are important for cell-type specific Col2a1 enhancer activity.

Naringin and naringenin are not the primary CYP3A inhibitors in grapefruit juice.

The effect of various citrus juices and solutions of naringin on CYP3A activity in rat liver microsomes was compared by measuring the formation of 6 beta-hydroxytestosterone from testosterone. Control enzyme activity was reduced by more than 70% by grapefruit juice. Freshly-squeezed sour (Seville) orange juice containing 20% as much naringin was comparable to grapefruit juice in its ability to inhibit microsomal activity. An aqueous solution of naringin at the same concentration as in grapefruit juice produced only a small decrease in 6 beta-hydroxytestosterone formation. Dilution of grapefruit juice with a naringin solution reduced the inhibitory activity of the juice even though naringin concentration did not change. Naringenin did not form under the incubation conditions used indicating that it did not contribute to the inhibition produced by grapefruit juice. Finally, organic extracts of grapefruit juice possessed considerable inhibitory activity even though naringin was not extractable. These data suggest that grapefruit juice inhibits CYP3A activity in vitro and that neither naringin nor naringenin are primarily responsible for this effect. A compound present in both grapefruit juice and Seville orange juice and extractable into organic solvents appears to be responsible.

Identification of a novel mutant transcript of laminin alpha 2 chain gene responsible for muscular dystrophy and dysmyelination in dy2J mice.

Murine dystrophia muscularis-2J (dy2J) is an autosomal recessive disorder characterized by muscular dystrophy and dysmyelination of peripheral nerve. Biochemical characterization of dy2J mice revealed the expression of a mutant laminin alpha 2 chain with a smaller molecular weight in the basal lamina of striated muscle and peripheral nerve. DNA sequencing of the alpha 2 chain cDNA amplified by RT-PCR from dy2J mice identified a novel and predominant transcript with a 171 base in-frame deletion. We also confirmed an underlying splice donor site mutation in the alpha 2 chain gene of the dy2J mouse. Translation of this variant transcript would result in the expression of a truncated alpha 2 chain having a 57 amino acid deletion (residues 34-90) and a substitution of Gln91Glu in the N-terminal domain VI, which is presumed to be involved in self-aggregation of laminin heterotrimers. Thus, the mutant alpha 2 chain could disrupt the formation of the laminin network and lead to muscle cell degeneration. Our results provide a molecular basis of muscular dystrophy and dysmyelination of peripheral nerve.

Cloning and expression of laminin alpha 2 chain (M-chain) in the mouse.

Laminins are a family of heterotrimeric glycoproteins specific to basement membranes. Laminin-2, consisting of alpha 2, beta 1 and gamma 1 chains, was originally identified in the basement membranes of skeletal muscle and peripheral nerve. We have isolated and sequenced the full-length cDNA for the mouse laminin alpha 2 chain. Four overlapping clones spanning 9,330 bp encode a predicted polypeptide of 3,106 amino acids having a calculated molecular mass of 390 kDa including a 23-amino-acid signal peptide. The amino acid sequence of the alpha 2 chain shares a 45.9% identify with that of the alpha 1 chain. Similar to the structure of the alpha 1 chain, the alpha 2 chain consists of several domains beginning at the N-terminus with three globular domains alternating with three epidermal growth factor-like domains followed by two alpha-helical domains and a C-terminal globular domain. The most N-terminal globular domain is highly conserved (77.3% identity) between the alpha 2 and alpha 1 chains, whereas the alpha-helical domains have low homology (30.3% identity). Northern blot and ribonuclease protection analysis revealed expression of mRNA for the alpha 2 chain in heart, kidney, liver, skin, lung and skeletal muscle of newborn mice. such a tissue distribution suggests a role for the alpha 2 chain and, consequently, laminin-2 or -4 not only in the organization and the function of nerve and muscle tissue but possibly also in the mesenchymal components of certain tissues.

A mammalian serine/threonine kinase receptor specifically binds BMP-2 and BMP-4.

Bone morphogenetic proteins (BMPs) are a class of related growth and differentiation factors within the TGF-beta superfamily of proteins which are known to induce cartilage and bone formation in adult animals and to be involved in many inductive events throughout embryonic development. Here we describe the molecular cloning and characterization of a mammalian receptor, CFK-43a, which specifically binds BMP-2 and BMP-4. This molecule is a member of the serine/threonine kinase receptor family which includes receptors for other TGF-beta superfamily members. CFK-43a binds other BMP family members with lower affinity, but does not bind TGF-beta. During embryogenesis, in situ hybridization analysis indicates that CFK-43a mRNA is localized in developing skeletal tissues in a complementary fashion to the transcripts for its ligands.

Deficiency of merosin in dystrophic dy mice and genetic linkage of laminin M chain gene to dy locus.

Merosin is the predominant laminin isoform in the basal lamina of striated muscle and peripheral nerve, and consists of M, B1 or S, and B2 chains. Here we have demonstrated that merosin is a native ligand for alpha-dystroglycan, an extracellular component of the dystrophin-glycoprotein complex. We have also mapped the mouse M chain gene, Lamm, to the same region of mouse chromosome 10 to which the dystrophia muscularis (dy) locus has been mapped. The dy mutation represents a severe neuromuscular disease resembling human muscular dystrophy. Analysis of merosin expression of dystrophic dy mice revealed a specific deficiency of merosin in skeletal muscle, cardiac muscle, and peripheral nerve. Our results indicate that merosin deficiency may be the primary defect in dy mice and suggest that a disruption of the link between alpha-dystroglycan and merosin may be involved in the pathogenesis of muscle degeneration and peripheral neuropathy in dy mice.

Regulation of expression of the chondrocytic phenotype in a skeletal cell line (CFK2) in vitro.

We have examined in vitro the spontaneous and regulated expression of phenotypic characteristics associated with differentiated chondrocytes in an established skeletal cell line (CFK2) derived from fetal rat calvariae. Extended culture of CFK2 cells resulted in the appearance of glycosaminoglycans and type II collagen in the cell layer in association with the formation of focal nodes of cells. In addition, induction of mRNA-encoding link protein, cartilage-specific proteoglycan core protein, and thrombospondin was observed in the differentiated population (dCFK2 cells). The expression of these mRNAs was present for at least two passages after subculturing the dCFK2 cells. The dCFK2 cells also demonstrated enhanced parathyroid hormone (PTH)-stimulated adenylate cyclase activity. Proliferation of CFK2 cells was stimulated by the peptide regulatory factors EGF and PTH and inhibited by the steroidal agents dexamethasone and retinoic acid. EGF and retinoic acid inhibited the formation of cell foci and glycosaminoglycan deposition and the expression of mRNA-encoding link protein. In contrast, PTH and dexamethasone enhanced the formation of focal cellular nodes and augmented matrix deposition and link protein mRNA expression. These studies therefore show that the CFK2 cell line can serve as a nontransformed model of rat chondrocytic cells in which both induction and regulation of the expression of cartilaginous matrix components can be observed. This line thereby provides a unique renewable source of chondrocytic precursor cells and an excellent in vitro model for evaluating temporal and environmental control of chondrocyte differentiation and cartilage matrix production.

Effect of protein and steroidal osteotropic agents on differentiation and epidermal growth factor-mediated growth of the CFK1 osseous cell line.

The effects of factors known to influence bone metabolism were examined using the osseous cell line CFK1. Parathyroid hormone (PTH) and dexamethasone (DEX) appeared to enhance the formation of cell foci of CFK1 cells in culture whereas retinoic acid (RA) caused a marked alteration in individual cell morphology. Bone morphogenetic protein (BMP-2) and PTH increased alkaline phosphatase activity, however, this index of differentiation was suppressed by epidermal growth factor (EGF), DEX, and RA. BMP-2 and EGF each stimulated DNA synthesis in a dose-dependent manner and enhanced cell numbers, but, no synergistic response of EGF and BMP-2 was observed. PTH and DEX failed to significantly alter cell number or EGF-stimulated DNA synthesis or cell proliferation. Although RA treatment of CFK1 cells resulted in a reduction in cell number compared to control, pretreatment with RA enhanced EGF-stimulated DNA synthesis and proliferative effects. At least part of this effect was by increasing the EGF receptor binding capacity of the cells. Furthermore, using cell cycle analysis, addition of EGF stimulated the progression of RA-treated cells into the DNA synthesis (S) phase with a reduced lag time. EGF and BMP-2, therefore, appear to exert a role in the expansion dynamics of the CFK1 population although BMP-2 may also enhance differentiation. PTH and DEX may act primarily to modulate the differentiated function of the CFK1 cells. RA inhibited cell proliferation and may mediate differentiation towards a less established cell population with upregulation of EGF receptors. The CFK1 cell model may, therefore, provide insight into microenvironmental control of growth and differentiation of precursor osseous cells.

Structural requirements for the growth factor activity of the amino-terminal domain of urokinase.

High molecular weight urokinase-type plasminogen activator (uPA) in which proteolytic activity was inactivated (diisopropyl fluorophosphate (DFP)-uPA), its amino-terminal fragment (ATF, amino acids (aa) 1-143), and fucosylated and defucosylated growth factor domains (GFD, aa 4-43) were tested for growth-promoting effects and binding in human SaOS-2 osteosarcoma cells and U-937 lymphoma cells. DFP-uPA, ATF, and both the fucosylated and defucosylated GFD were capable of competing with 125I-ATF for binding to both SaOS-2 and U-937 cells. DFP-uPA, ATF, and fucosylated GFD were also mitogenic in SaOS-2 cells and increased cell numbers. However, defucosylated GFD was nonmitogenic in SaOS-2 cells and did not stimulate cell proliferation, even though it bound to these cells in a manner equivalent to the fucosylated GFD. A nonglycosylated high molecular weight uPA expressed and purified from Escherichia coli inhibited 125I-ATF binding to SaOS-2 cells but was also nonmitogenic. No mitogenic activity was observed in U-937 cells treated with the uPA forms capable of eliciting a mitogenic response in SaOS-2 cells. Proteolytically prepared kringle domain (aa 47-135) and low molecular weight uPA (aa 144-411) did not compete for 125I-ATF binding and did not elicit any mitogenic response in either of the cell lines tested. In addition, tissue plasminogen activator (tPA), which has been shown to be homologous to uPA in its growth factor domain and is also fucosylated, did not inhibit 125I-ATF binding nor elicit any mitogenic response. These results demonstrate that the GFD, implicated in binding to the uPA receptor, is also responsible for growth factor like activity in SaOS-2 cells and that the fucosylation at Thr18 within this domain may serve as a molecular trigger in eliciting this response.

Enhanced growth of a human keratinocyte cell line induced by antisense RNA for parathyroid hormone-related peptide.

We have used antisense RNA technology to inhibit endogenous parathyroid hormone-related peptide (PTHRP) production in the established human keratinocyte cell line, HPK1A, in order to assess the role of PTHRP as a potential modulator of cell growth. Rat PTHRP cDNA was cloned into the replication defective retroviral vector pYN in an antisense orientation and a stable cell line (HPK1A-AS) was generated after infection by amphotropic virus and selection by the neomycin derivative, G418. Expression of the transfected antisense sequence was confirmed with an RNA sense probe for PTHRP. The effect of the retrovirally mediated gene transfer on the endogenous PTHRP transcript was examined with an RNA antisense probe which demonstrated an absence of the endogenous transcript in HPK1A-AS cells. A 1.6-kilobase transcript was, however, present in equivalent quantities in both uninfected HPK1A and pYN-infected (HPK1A-pYN) cells. Immunocytochemistry and assessment of PTHRP secretion into the medium using an NH2-terminal radioimmunoassay and a UMR 106 adenylate cyclase bioassay confirmed the absence of PTHRP in HPK1A-AS cells. Examination of the inhibition of PTHRP production on cell growth demonstrated a reduction in doubling time and an increase in [3H]thymidine incorporation. Cell cycle analysis showed an increase in the proportion of the cell population in the S phase (relative to G0/G1) in HPK1A-AS cells compared to HPK1A or HPK1A-pYN cells. These data, therefore, indicate that endogenous PTHRP acts as an effective inhibitor of cell growth in this keratinocyte model and that this action occurs, at least in part, by diminishing entry into the S phase of the cell cycle. Furthermore, the antisense RNA method is a potent one to evaluate the cellular actions of PTHRP.

Heterogeneous intracellular free calcium responses to parathyroid hormone correlate with morphology and receptor distribution in osteogenic sarcoma cells.

The osteogenic sarcoma cell line UMR 106-01 exhibits heterogeneous morphology and hormone response in subconfluent monolayer cultures. In these studies we have explored the correlation between morphological profiles and patterns of cytosolic calcium [Ca2+]i response to PTH and other agonists in single UMR 106-01 cells loaded with the Ca(2+)-sensitive fluorescent indicator, fura-2. Realtime recording of [Ca2+]i revealed that PTH (10(-7) M) produced a transient [Ca2+]i rise in 19% of the cells studied. [Ca2+]i transients were also induced by prostaglandins E2 and F2 alpha, and fetal bovine serum, but with different response frequencies (20%, 12%, and 58%, respectively). Spatial resolution of changes in [Ca2+]i by video image analysis revealed that the response to PTH was more frequent in large polygonal cells with long cytoplasmic processes and less common in smaller cells growing in clusters, whereas there was no clear subtype specificity for the effects of epidermal growth factor and fetal bovine serum on [Ca2+]i. Autoradiographic analysis of cell monolayers demonstrated a higher density of PTH-binding sites on cells with cytoplasmic extensions, whereas epidermal growth factor-binding sites were largely on colony-forming cells. Thus, the [Ca2+]i response to hormonal stimulation is heterogeneous within UMR 106-01 cell populations and within single cells, and it correlates with receptor density. This suggests that osteoblastic cells respond to PTH by activation of changes in [Ca2+]i only at certain specific steps during osteoblast development or stages of the cell cycle.

Biochemical and morphological analysis of the interaction of epidermal growth factor and parathyroid hormone with UMR 106 osteosarcoma cells.

The interaction of epidermal growth factor (EGF) and PTH with UMR 106 osteosarcoma cells was examined biochemically and morphologically. EGF inhibited PTH-stimulated adenylate cyclase activity in association with a reduction in PTH receptors and a decrease in the activity of the stimulatory guanyl nucleotide-binding protein (Gs). Universal inhibition of agonist-stimulated adenylate cyclase activity did not occur, inasmuch as EGF did not reduce prostaglandin E2-enhanced enzyme activity. The influence of EGF on PTH action correlated with its effect in the UMR 106 cell population of promoting entry into the cell cycle. Morphological analysis with radioautography indicated that both EGF and PTH receptors could be colocalized to certain UMR 106 cells, but that each were more abundantly distributed over discrete UMR 106 cell types. Based on the distribution of [3H]thymidine incorporation, EGF receptors were predominantly found over rapidly proliferating cells, whereas PTH receptors were most densely distributed over more quiescent cells. The results indicate that EGF and PTH receptors are localized over specific types within the heterogeneous population of UMR 106 cells and suggest that EGF may reduce PTH action in these cultures by enhancing the proliferation of progenitor cells lacking PTH receptors and reducing differentiation in this cell population, which leads to PTH receptor-enriched target cells. EGF and PTH receptors may, therefore, be useful as probes to examine both functional interactions and differentiation pathways among cells in osteoblast models in vitro and perhaps in vivo.

Biological properties of synthetic human parathyroid hormone: effect of deamidation at position 76 on agonist and antagonist activity.

Recent studies have suggested a role for the carboxyl-terminus of PTH in the binding of the molecule to renal and skeletal receptors, but the functional significance of this binding remains uncertain. We have investigated the possible role of this region by examining the effect of substituting the asparagine residue at position 76 of the native human molecule [Asn76]hPTH-(1-84) with an aspartate residue, [Asp76] hPTH-(1-84) on activity in both renal and skeletal cytochemical (CBA) and adenylate cyclase (AC) bioassays. In the renal CBA, [Asp76]hPTH-(1-84) was considerably less potent than [Asn76]hPTH-(1-84) and produced dose-dependent inhibition of the bioactivity of intact bovine (b) PTH-(1-84), bPTH-(1-34), and [Asn76]hPTH-(1-84). [Asp76]hPTH-(39-84) inhibited the response to intact PTH to a lesser extent, whereas [Asp76]hPTH-(53-84) had no antagonistic activity. In the metatarsal CBA, [Asp76]hPTH-(1-84) inhibited the response to intact PTH, but was less potent than in the renal CBA. In both renal (OK) and skeletal (UMR) cell AC assays [Asp76]hPTH-(1-84) and [Asn76]hPTH-(1-84) were equipotent agonists. Therefore, the CBAs are much more sensitive to modification of the carboxyl end of the molecule than AC assays. The antagonist properties of [Asp76]hPTH-(1-84) appeared to be mediated by phosphodiesterase activation as theophylline abolished the antagonism of this analog. These studies indicate that generation of PTH analogs, modified at the carboxyl-terminal region as well as at the amino-terminus, may be useful for developing potent PTH antagonists.

Establishment of an osseous cell line from fetal rat calvaria using an immunocytolytic method of cell selection: characterization of the cell line and of derived clones.

Using selective media and complement-mediated lysis of primary cultures of a fetal rat calvarial cell population, we have developed a cell line (OBCK6) that exhibits osteoblastic characteristics. OBCK6 cells demonstrated enhanced parathyroid hormone (PTH)-stimulated adenylate cyclase activity relative to the primary calvarial population, production of alkaline phosphatase activity and type 1 collagen, and the capacity to form mineralized nodules in unsupplemented medium after prolonged (22-26 day) culture. Two sublines, CFK1 and CFK2, which were isolated by dilution cloning, differed morphologically and with respect to growth rate. CFK1 cells demonstrated high PTH and prostaglandin E2-stimulated adenylate cyclase activity, whereas only low PTH-stimulated activity was observed in CFK2 cells. Retinoic acid and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] each reduced PTH-stimulated adenylate cyclase activity in both the cell types. Retinoic acid and dexamethasone reduced and 1,25(OH)2D3 enhanced alkaline phosphatase activity in these cells. PTH significantly augmented alkaline phosphatase activity to a much greater extent in CFK1 than in CFK2 cells. Both CFK1 and CFK2 cells expressed type I but type III collagen, and neither expressed osteocalcin. Strong Alcian blue staining of CFK2 cells was suggestive of a cartilaginous phenotype. These three cell lines, therefore, demonstrated discrete characteristics of skeletal cell function and should provide important models for evaluation of mechanisms of mineralization and for control of skeletal cell growth and mesenchymal differentiation in vitro.

Synthesis and characterization of extended and deleted recombinant analogues of parathyroid hormone-(1-84): correlation of peptide structure with function.

Recombinant analogues of human parathyroid hormone [hPTH-(1-84)] were expressed in Escherichia coli harboring plasmids containing synthetic genes under the control of the lac promoter. The level of expression of the gene encoding the truncated analogue, hPTH-(3-84), was greater than that of the gene encoding full-length hPTH-(1-84) but less than that of the gene encoding proparathyroid hormone (hProPTH). This may be due in part to the relative efficiency of translation of the mRNA as suggested by secondary structure analysis and in part because of enhanced stability of the extended peptide. Formylmethionyl derivatives of hProPTH and of hPTH-(3-84) and underivatized hPTH-(3-84) were purified by HPLC, and their identity was confirmed by NH2-terminal sequencing and amino acid analysis. The bioactivity of these recombinant peptides was then tested in skeletal and renal adenylate cyclase assays in vitro and in assays examining effects on plasma and urine calcium and phosphate levels and on urine cyclic AMP levels in vivo. The NH2-terminally extended analogue fMet-hProPTH displayed 10% of the in vitro activity of hPTH-(1-84) and was a partial agonist in vivo. The peptides hPTH-(3-84) and fMet-hPTH-(3-84) were inert in vitro and were very weak in vitro antagonists when compared to the NH2-terminal analogue bovine [Nle8,18Tyr34]PTH-(3-34)-NH2. In vivo, hPTH-(3-84) and the bPTH-(3-34) analogue, when assayed at a 10:1 molar ratio relative to bPTH-(1-84), were each inert, and neither demonstrated antagonist activity at these concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)