Characterization of Gas6, a member of the superfamily of G domain-containing proteins, as a ligand for Rse and Axl.

Rse, Ax1, and c-Mer comprise a family of cell adhesion molecule-related tyrosine kinase receptors. Human Gas6 was recently shown to act as a ligand for both human Rse (Godowski et al., 1995) and human Ax1 (Varnum et al., 1995). Gas6 contains an NH2-terminal Gla domain followed by four epidermal growth factor-like repeats and tandem globular (G) domains. The G domains are related to those found in sex hormone-binding globulin and to those utilized by laminin and agrin for binding to the dystroglycan complex. A series of Gas6 variants were tested for their ability to bind to Rse and Ax1. The Gla domain and epidermal growth factor-like repeats were not required for receptor binding, as deletion variants of Gas6 which lacked these domains bound to the extracellular domains of both Rse and Axl. A deletion variant of Gas6 containing just the G domain region was shown to activate Rse phosphorylation. These results provide evidence that G domains can act as signaling molecules by activating transmembrane receptor tyrosine kinases. Furthermore, they provide a structural link between the activation of cell adhesion related receptors and the control of cell growth and differentiation by the G domain-containing superfamily of proteins.

In vitro characterization of four novel classes of growth hormone-releasing peptide.

Reexamination of the hexapeptide GH-releasing peptide (GHRP-6) structure/function has lead to the development of four novel classes of compound that stimulate GH release. Each class is represented as follows: a pentapeptide, G-7039; a tetrapeptide, G-7134; a pseudotripeptide, G-7502; and a rigid cyclic heptapeptide, G-7203. The EC50 values for these compounds, determined by GH dose-response curves using primary cultures of rat pituitary cells, were 0.18, 0.34, 10.6, and 0.43 nM, respectively. To demonstrate that these compounds were acting at the putative GHRP receptor, challenges were made using combinations that included GHRP-6 and GH-releasing hormone (GHRH). All four new classes further increased GH release in combination with GHRH, but not with GHRP-6. Homologous desensitization occurred after 45 min of exposure to the new compounds while the cells remained sensitive to GHRH. Somatostatin inhibited all of these compounds. Additionally, G-7039 elevated free calcium, as occurs with GHRP-6. All four classes elicited a robust GH release, a small increase in PRL, and no change in LH, FSH, ACTH, or TSH. We conclude that these novel compounds are potent and direct stimulators of pituitary GH release, with in vitro attributes that suggest mediation via a specific GHRP-like mechanism.

Growth hormone secretagogues: characterization, efficacy, and minimal bioactive conformation.

Another class of growth hormone (GH) secretagogues has been discovered by altering the backbone structure of a flexible linear GH-releasing peptide (GHRP). In vitro and in vivo characterization confirms these GH secretagogues as the most potent and smallest (M(r) < 500) reported. Anabolic efficacy is demonstrated in rodents with intermittent delivery. A convergent model of the bioactive conformation of GHRPs is developed and is supported by the NMR structure of a highly potent cyclic analog of GHRP-2. The model and functional data provide a logical framework for the further design of low-molecular weight secretagogues and illustrate the utility of an interdisciplinary approach to elucidating potential bound-state conformations of flexible peptide ligands.

Effects of kistrin on bone resorption in vitro and serum calcium in vivo.

In many cell systems, cell-cell and cell-matrix interactions are mediated by integrins, a family of cell surface heterodimeric glycoprotein receptors. Osteoclast integrins may play a role in the process of bone resorption. Osteoclasts express the alpha v and beta 3 subunits of the vitronectin receptor and adhere to a wide range of proteins in vitro, all which contain the amino acid sequence Arg-Gly-Asp (RGD), an adhesion site recognition sequence common to many protein ligands that bind to integrins. The effect of kistrin, an RGD-containing snake venom protein, on osteoclast-mediated bone resorption was investigated in vivo and in vitro. When kistrin was infused into normocalcemic and hypercalcemic mice, serum calcium was significantly lowered at 3 and 6 h after the start of infusion, indicating an inhibitory effect on osteoclast activity in vivo. In vitro, kistrin potently inhibited bone resorption by isolated rat osteoclasts cultured on slices of bovine bone, and kistrin also inhibited the attachment of 293 cells expressing recombinant human alpha v beta 3 to fibrinogen (IC50 = 1 nM). These results indicate the potential therapeutic use of RGD-containing molecules for hypercalcemia of malignancy or for other disorders associated with bone loss.

Differential effects of transforming growth factor-beta 1 and bone morphogenetic protein 4 on gene expression and differentiated function of preosteoblasts.

Transforming growth factor-beta (TGF-beta) and bone morphogenetic protein 4 (BMP 4) are both able, under certain circumstances, to induce endochondral bone formation in vivo. This study compared the effects of TGF-beta 1 and BMP 4 on the gene expression of a retinoic acid (RA) responsive rat clonal preosteoblast cell line, UMR 201, as well as the way in which these proteins interact with RA in these cells. Both similarities as well as differences between the effects and mechanism of action of TGF-beta 1 and BMP 4 were demonstrated. TGF-beta 1 (0.1 ng/ml) strongly induced matrix gla protein (MGP) mRNA and increased the steady state osteonectin (ON) mRNA level. Cotreatment with TGF-beta 1 and RA did not result in a further increase in MGP mRNA expression. In contrast, BMP 4 alone had no influence on MGP or ON mRNA expression but it significantly enhanced the RA induction of MGP mRNA. Pro-alpha 1(l) collagen mRNA was increased by TGF-beta 1 (1 ng/ml) and BMP 4 (50 ng/ml). The addition of either TGF-beta 1 or BMP 4 together with RA resulted in a further increase in pro-alpha 1 (l) collagen mRNA levels. Both RA and TGF-beta 1, but not BMP 4, increased the transcriptional rate of the pro-alpha 1 (l) collagen gene. TGF-beta 1 reduced the constitutive as well as RA-induced expression of osteopontin (OP) mRNA while BMP 4 reduced only the constitutive expression of OP mRNA. RA increased the transcriptional rate of the OP gene. Since the responses of UMR 201 cells to these structurally related factors were not identical, the results lend support to the concept that the coordinated expression of members of the TGF-beta 1 superfamily may be necessary to control the progression of specific cell types through their differentiation pathways.

Natural bovine osteogenin and recombinant human bone morphogenetic protein-2B are equipotent in the maintenance of proteoglycans in bovine articular cartilage explant cultures.

Osteogenin and related bone morphogenetic proteins are members of the transforming growth factor-beta superfamily, and were isolated by their ability to induce cartilage and bone formation in vivo. The influence of osteogenin, purified from bovine bone, and of recombinant human bone morphogenetic protein-2B (BMP-2B) has been examined in bovine articular cartilage explants. Both differentiation factors stimulated in a dose-dependent manner the synthesis of proteoglycans and decreased their rate of degradation. At a dose of 30 ng/ml, proteoglycan synthesis was increased to levels observed with either 20 ng/ml insulin-like growth factor I, 10 ng/ml transforming growth factor-beta, or 20% fetal bovine serum. This increase of biosynthetic rates above basal medium levels was observed in young, adolescent, and adult tissues. Analysis of the size of the newly synthesized proteoglycans, the glycosaminoglycan chain size, and the glycosaminoglycan type of explants treated with osteogenin or BMP-2B were very comparable to each other, and to proteoglycans isolated from cartilage treated with either insulin-like growth factor I or fetal bovine serum. These results demonstrate that osteogenin and BMP-2B alone are capable of stimulating and maintaining the chondrocyte phenotype in vitro.

Bone morphogenetic protein-2b stimulation of growth and osteogenic phenotypes in rat osteoblast-like cells: comparison with TGF-beta 1.

The biologic effects of recombinant human bone morphogenetic protein-2b (BMP-2b = BMP-4) were studied and compared with transforming growth factor-beta 1 (TGF-beta 1) in fetal rat osteoblast-like (ROB) cells. Similar to the effects of TGF-beta 1, BMP-2b stimulated DNA and collagen synthesis as well as protein accumulation. Unlike TGF-beta 1, which inhibited alkaline phosphatase activity, BMP-2b enhanced enzyme activity eight-to ninefold over the control level. The present study demonstrates direct actions of BMP-2b on bone-associated cells to stimulate osteogenic phenotypes in vitro and provides a cellular mechanism for the induction of bone formation by BMP-2b in vivo.

A potent inhibitor of osteoclastic bone resorption within a highly conserved pentapeptide region of parathyroid hormone-related protein; PTHrP[107-111].

We have recently shown that a carboxyl-terminal sequence of parathyroid hormone-related protein, PTHrP[107-139], is a potent direct inhibitor of osteoclastic bone resorption. We now report that this bone resorption inhibitory activity, which we have called osteostatin, is entirely contained within the highly conserved pentapeptide PTHrP[107-111]. Our results indicate that processing at residue 106 and a free amino terminus is required for full activity of the peptide. The retroinverted peptide is considerably less potent than the parent peptide. The retention of full biological activity in such a short fragment was unexpected. This data provides the basis for the development of further analogs with potential therapeutic use in disorders associated with increased osteoclastic bone resorption.

Stimulation of chondrogenesis in limb bud mesoderm cells by recombinant human bone morphogenetic protein 2B (BMP-2B) and modulation by transforming growth factor beta 1 and beta 2.

Bone morphogenetic protein 2B (BMP-2B) also called BMP-4 is one of a family of cartilage and bone-inductive proteins derived from bone matrix and belongs to the transforming growth factor beta (TGF-beta) superfamily. These bone-inductive proteins isolated from adult bone may be involved in bone repair. However, they may also play a role in cartilage and bone formation during embryonic development. To test whether BMP-2B influences cartilage formation by embryonic cells, recombinant human BMP-2B was applied to cultured limb bud mesoderm plated at three different densities. BMP-2B stimulated cartilage formation as assessed by Alcian blue staining and incorporation of radioactive sulfate into sulfated proteoglycans. Cells cultured at all three densities in the presence of 10 ng/ml BMP-2B formed a nearly continuous sheet of cartilage with abundant extracellular matrix and type II collagen. In addition, when cells were cultured in 0.5% serum in the presence of 10 ng/ml of BMP-2B for 5 days there was an increase in alkaline phosphatase as detected by histochemical and biochemical methods. Transforming growth factor beta isoforms (TGF-beta 1 and TGF-beta 2) inhibited sulfate incorporation into proteoglycans in a dose-dependent manner. This inhibition by TGF beta was overcome by recombinant BMP-2B. This study demonstrates that recombinant BMP-2B stimulates cartilage formation by chick limb bud mesoderm in vitro and is further modulated by TGF-beta isoforms.

Retinoic acid modulation of mRNA levels in malignant, nontransformed, and immortalized osteoblasts.

Clonal cell lines presumably "arrested" at a particular stage of differentiation are useful models to study the processes of differentiation in osteoblasts. UMR-201 is a presumptive preosteoblastic nontransformed rat clonal cell line with a limited life span in culture. Two immortalized cell lines, UMR-201-10A (10A) and UMR-201-10B (10B), were derived from UMR-201 by stable transfection with simian virus (SV) 40 large T antigen. This study compares the growth and profile of gene expression of the immortalized cell lines with those of UMR-201 and UMR-106-06, a rat clonal cell line with well-defined osteoblast-like phenotypic characteristics. All four cell lines constitutively expressed the mRNA for the gamma, alpha, and beta receptors for retinoic acid (RA), the growth hormone receptor, pro-alpha 1(I) collagen, osteonectin, bone proteoglycan I, and bone morphogenetic proteins (BMP) 1 and 2A. Alkaline phosphatase mRNA was absent in the preosteoblast cell lines but was induced by treatment with 10(-6) M RA, which also increased the steady-state levels of mRNA for osteopontin and BMP1. mRNA for matrix gla protein was constitutively present and further induced by RA in UMR-201 and 10B only. Messenger RNA for bone sialoprotein and bone morphogenetic protein 3 were constitutively expressed in UMR-106-06 and UMR-201 but absent in the immortalized cell lines. None of the cell lines expressed measurable mRNA for bone gla protein or bone proteoglycan II. 10B grew more rapidly than UMR-201, but unlike UMR-201, it was also able to proliferate in serum-free medium and exhibit anchorage-independent growth. In summary, this study identifies novel retinoic acid effects on gene expression in these cells. Differences noted in the expression of mRNAs between UMR-106-06 and the other cell lines may provide some insight into the sequence of expression of these phenotypic characteristics as osteoblasts differentiate.

Identification and characterization of cellular binding proteins (receptors) for recombinant human bone morphogenetic protein 2B, an initiator of bone differentiation cascade.

Bone morphogenetic protein 2B (BMP 2B), is a heparin-binding bone differentiation factor that initiates endochondral bone formation in rats when implanted subcutaneously. The molecular mechanism of action of this differentiation factor is not known, and as a first step we have examined BMP 2B-responsive cells for the presence of specific cellular binding proteins. Using 125I-labeled BMP 2B, specific high-affinity binding sites for recombinant human BMP 2B on MC3T3 E1 osteoblast-like cells as well as on NIH 3T3 fibroblasts were identified. Platelet-derived growth factor, insulin-like growth factor 1, basic fibroblast growth factor, epidermal growth factor, and transforming growth factor beta did not compete for the binding of radiolabeled BMP 2B. The binding of BMP 2B is a time- and temperature-dependent process. Chemical crosslinking of radiolabeled BMP showed two components (apparent size, 200 and 70 kDa in MC3T3 E1 cells and 200 and 90 kDa in NIH 3T3 cells). A minor component at 60 kDa was also detected in both cell lines. Scatchard analysis of the binding data showed a high-affinity receptor with an apparent dissociation constant of 128 +/- 40 pM in MC3T3 E1 cells. These data demonstrate specific, high-affinity cell-surface binding proteins for BMP 2B.

Bone-inducing activity of mature BMP-2b produced from a hybrid BMP-2a/2b precursor.

The human osteoinductive proteins BMP-2a and BMP-2b have been cloned and expressed in mammalian cells. In order to improve expression levels we examined the role of the proregion in assembly and export. Use of the BMP-2a proregion combined with the mature region of BMP-2b leads to dramatically improved expression of mature BMP-2b. Mature BMP-2b has been purified to near homogeneity from the BMP-2a/2b hybrid, and its structural properties and biological activity determined. Recombinant mature BMP-2b homodimer elicits bone formation in vivo.

Fetal magnesium homeostasis in the sheep.

The fetal plasma magnesium concentration exceeds that of the mother but the difference is small compared to that of ionized calcium concentration. Although not fully independent of changes in maternal magnesaemia, fetal magnesaemia showed a high degree of autonomy during both hypermagnesaemic and hypomagnesaemic changes induced in the ewe. As with calcium, the placental gradient is reversed after fetal thyroparathyroidectomy (TXPTX) with thyroxine replacement. During perfusion in situ of the placenta from such TXPTX fetuses isolated from the fetus itself, a stable positive placental gradient of magnesium concentration could be re-established between the perfusing blood and the maternal circulation. As with calcium, this gradient could be increased by fetal calf parathyroid extract, parathyroid hormone-related protein (PTHrP 1-141), PTHrP (1-84) but not by PTHrP (1-34). It was concluded that a mid-molecule portion of PTHrP can stimulate a putative placental pump which is responsible for the gradients of both calcium ions and magnesium across the ovine placenta.

Purification and characterization of recombinant human parathyroid hormone-related protein.

Full-length human parathyroid hormone-related protein (PTHrP-(1-141] as well as a carboxyl-terminal shortened form (PTHrP-(1-108] have been expressed from recombinant DNA-derived clones. These proteins were expressed in Escherichia coli as fusion proteins so that cyanogen bromide cleavage yields the desired product. Both proteins were purified and then characterized by sodium dodecyl sulfate gel electrophoresis, amino-terminal amino acid sequencing, peptide mapping, and mass spectral analysis. Recombinant PTHrP-(1-141), PTHrP-(1-108), synthetic PTHrP-(1-34), and naturally derived PTHrP are all equipotent in the stimulation of cyclic AMP levels in the osteoblast-like cell line UMR 106-01. However, PTHrP-(1-141) and -(1-108) are two to four times more active than PTHrP-(1-34) in the stimulation of plasminogen activator activity from this cell line. PTHrP-(1-141) reacts equipotently with PTHrP-(1-34) in a radioimmunoassay using an antiserum prepared against PTHrP-(1-34). PTHrP-(1-141), -(1-108), and -(1-84) were used as PTHrP-specific mobility standards on sodium dodecyl sulfate gel electrophoresis to determine the approximate length of two forms of naturally derived PTHrP. The data show that PTHrP purified from the lung tumor cell line BEN contains a major form of about 108 amino acids and another form of about 141 amino acids.

Purification and partial amino acid sequence of osteogenin, a protein initiating bone differentiation.

Osteogenin was purified from bovine bone matrix and its activity monitored by an in vivo bone induction assay. The purification method utilized extraction of the bone-inducing activity with 6 M urea, followed by chromatography on heparin-Sepharose, hydroxyapatite, and Sephacryl S-200. Active fractions were further purified by preparative sodium dodecyl sulfate gel electrophoresis without reduction. Osteogenin activity was localized in a zone between 30 and 40 kDa. The amino acid sequences of a number of tryptic peptides of the gel-eluted material were determined. Reduction and alkylation of purified osteogenin in 7 M guanidine hydrochloride resulted in the total loss of biological activity. Sodium dodecyl sulfate gel electrophoresis under reducing conditions revealed a broad band with an apparent molecular mass of 22 kDa.

Stimulation of ovine placental calcium transport by purified natural and recombinant parathyroid hormone-related protein (PTHrP) preparations.

Perfusion in situ of the placenta of previously thyroparathyroidectomized fetal lambs has been used to compare the ability of various forms of parathyroid hormone-related protein (PTHrP) to stimulate placental calcium transport. Whereas PTHrP (1-34) was without effect, PTHrP (1-141) was active but usually after a delay of up to 1 h, in common with the effect noted when using extracts of fetal parathyroid glands. In contrast, PTHrP (1-84) and PTHrP (1-108), tended to show a more rapid stimulatory action. It is suggested that post-translational processing of PTHrP (1-141) may occur as an activating step in the placenta in vivo.