Serum and dialysate osteocalcin levels in hemodialysis and peritoneal dialysis patients and after renal transplantation

Serum osteocalcin (BGP), a vitamin K-dependent gamma-carboxyglutamic acid (GLA) containing bone protein, provides an index of bone turnover in patients with a variety of metabolic bone diseases. BGP increases with increasing age in both sexes, but more so in women. BGP rises above normal when the glomerular filtration rate falls below 30 ml/min. Because of its importance in bone disease, its low mol wt, and the effect of uremia, we measured BGP by RIA in serum and dialysate fluid in patients on hemodialysis (HD) or peritoneal dialysis (PD). In 32 HD patients (22 women and 10 men), serum BGP was not different pre- and postdialysis [67.5 +/- 4.4 (+/- SEM) ng/ml vs. 67.7 +/- 5.2), but was significantly elevated compared to the level in normal subjects (7.3 +/- 0.8 ng/ml). The sex difference previously reported in normal subjects was not found in patients with renal failure. The serum BGP level in 8 PD patients was 49.4 +/- 6.9 ng/ml, with a peritoneal fluid concentration of 27.6 +/- 9.3 ng/ml. The hemodialysate fluid concentration of BGP was 1.7 +/- 0.4 ng/ml, which was significantly lower than the serum BGP levels in the HD patients, the PD patients, and peritoneal fluid (P less than 0.01). A significant correlation existed among BGP, alkaline phosphatase, immunoreactive PTH, creatinine, and blood urea nitrogen. We conclude that BGP is markedly elevated in patients with renal failure, not altered in the serum by HD or PD, but very low in HD dialysate fluid. These findings may reflect a combination of impaired clearance and increased skeletal production. The difference in clearance between the peritoneal and hemodialysis fluid is compatible with the mol wt of BGP. In 15 patients who had successful kidney transplantation, serum BGP was normal despite an elevated serum PTH level.     

Tumor necrosis factor-alpha inhibits 1,25-dihydroxyvitamin D3-stimulated bone Gla protein synthesis in rat osteosarcoma cells (ROS 17/2.8) by a pretranslational mechanism

Tumor necrosis factor-alpha (TNF alpha), a 17,000 mol wt protein, mediates a variety of immunological and inflammatory events. TNF alpha is a potent inhibitor of bone collagen synthesis and stimulator of osteoclastic bone resorption, the net effect of which is to cause bone loss. We have previously reported that TNF alpha inhibits the synthesis of collagen by osteoblastic cells in culture out of proportion to effects on total protein synthesis, suggesting that inhibition of bone formation by TNF alpha may be due to selective inhibition of matrix protein synthesis. To further test this hypothesis and to evaluate the mechanism of TNF alpha action, we studied the effect of TNF alpha on synthesis of the osteoblast-specific bone Gla protein (BGP) by ROS 17/2.8 cells, which have the osteoblast phenotype. Cells were cultured with 10 nM 1,25-dihydroxyvitamin D3 to stimulate BGP secretion, followed by the addition of TNF alpha (1-100 ng/ml) in 1,25-dihydroxyvitamin D3-containing medium. TNF alpha (10 ng/ml) inhibited BGP secretion to 42 +/- 5%, 19 +/- 10%, and 15 +/- 3% of control values after 24, 48, and 72 h of treatment. After 48 h, inhibition of BGP secretion was observed with 2 ng/ml TNF alpha and was maximum with 100 ng/ml. To determine the effect of TNF alpha on total protein synthesis, cells were pulse labeled with [14C]leucine during the last 4 h of TNF alpha treatment, and incorporation of radioactivity into trichloroacetic acid-precipitable protein in cell layer and medium was determined. The TNF alpha inhibition of BGP secretion was independent of changes in [14C]leucine incorporation, suggesting that TNF alpha did not have a general inhibitory effect on total protein synthesis. Cell number was not affected by TNF alpha. Northern analysis of steady state BGP mRNA revealed a dose-dependent decrease in the BGP/cyclophilin mRNA hybridization signal intensity after 24 h of treatment. The maximum inhibitory effect was 41 +/- 5% of the control value with 100 ng/ml TNF alpha. The effect of TNF alpha on steady state BGP mRNA levels was not prevented by treatment of cells with cycloheximide, suggesting that TNF-induced new protein synthesis was not required for TNF alpha action. These results suggest that the mechanism of TNF alpha inhibition of BGP synthesis includes a pretranslational site and support the hypothesis that TNF alpha inhibits bone formation by a selective inhibition of matrix protein production.     

Biosynthesis of a novel thyroxine-binding protein (27K protein) in human hepatoma (Hep G2) cells

Human hepatoma (Hep G2) cells were shown to synthesize and secrete a novel T4-binding protein, called 27K protein for its apparent mol wt on sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The mRNA coding for this protein was characterized by immunoprecipitation of [125I]T4 bound to 27K protein secreted into the medium of oocytes injected with total Hep G2 RNA. Sucrose gradient fractionation of RNA from Hep G2 cells showed that TBG mRNA and 27K mRNA had different sizes, indicating that TBG and 27K protein are two distinct proteins. In vitro translation of RNA in a rabbit reticulocyte lysate demonstrated that the translation product immunoprecipitated by anti-27K serum had the same mol wt as the immunoprecipitated protein from whole cells labeled with [35S]methionine, thus suggesting that 27K protein is neither derived from TBG nor synthesized through a larger mol wt precursor, and also that it does not contain carbohydrates. The absence of carbohydrates was further supported by the observation that [3H]mannose was not covalently bound to the 27K protein when Hep G2 cells were labeled with [3H]mannose, nor was there a shift in apparent mol wt when the cells were treated with the glycosylation inhibitor tunicamycin. The kinetics of secretion of 27K protein were similar to those of albumin and faster than those of TBG, which is also in keeping with the nonglycoprotein nature of 27K protein.     

The propeptide of rat bone gamma-carboxyglutamic acid protein shares homology with other vitamin K-dependent protein precursors.

The molecular cloning of bone gamma-carboxyglutamic acid (Gla) protein (BGP; osteocalcin) was accomplished by constructing a phage lambda gt11 cDNA library from the rat osteosarcoma cell line ROS 17/2 and screening this library with antibodies raised against BGP from rat bone. By sequencing several cloned cDNAs, we have established a 489-base-pair sequence that predicts a mature BGP of 50 amino acid residues with an NH2-terminal extension of 49 residues. The leader peptide consists of a hydrophobic signal peptide followed by a basic propeptide of 26 or 27 residues that is cleaved after an Arg-Arg dipeptide prior to secretion from the cell. Mature rat BGP is extremely homologous to BGPs from other species except for its COOH-terminal sequence. A stretch of 9 residues proximal to the NH2 terminus of secreted BGP is strikingly similar to the corresponding regions in known propeptides of the gamma-carboxyglutamic acid-containing blood coagulation factors. We suggest that this common structural feature may be involved in the posttranslational targeting of these polypeptides for vitamin K-dependent gamma-carboxylation.     

Radioimmunoassay for the vitamin K-dependent protein of bone and its discovery in plasma.

The vitamin K-dependent protein of bone has been detected in human plasma by radioimmunoassay at 4.5 ng per ml. The plasma protein has the same apparent molecular weight as the pure bone Gla protein (BGP) and other studies indicate the plasma protein is probably the intact bone protein. BGP also has been detected in bovine serum by radioimmunoassay. The bovine serum levels of BGP decrease with developmental age from 200 ng per ml in fetal calves to 26 ng per ml in adult cows. The implications of the discovery of BGP in plasma to the function of this unique protein are discussed. This assay employs rabbit antibody directed against calf BGP and has a sensitivity of 0.1 ng. The antibody crossreacts with purified human BGP but not with BGP from rat or rabbit bone. Studies with peptides of known structure derived from enzymatic digests of BGP indicate that the rabbit antibody recognizes the COOH-terminal region of the 49-residue calf bone protein.     

Mononuclear cells are not involved in BGP synthesis and secretion

Summary Osteocalcin or bone GLA protein (BGP) is found at high levels in only two tissues, the extracellular matrix of bone and dentine. Tissue culture experiments have demonstrated that BGP is synthesized by two osteoblastic osteosarcoma cell lines (ROS 2/3 and 17/2) and by normal osteoblastic cells in primary culture. BGP was not found in rat cartilage nor in liver, kidney, lung, spleen, brain, heart, thymus, skeletal muscle. In this study secretion of BGP was assayed by RIA in the supernatants of 48-hour cultures of peripheral blood lymphocytes or monocytes. Lymphocyte cultures were carried out using RPMI-1640 supplemented with L-glutamine and antibiotics at the concentration of 1×10⁶ cells/ml and activated by PHA (10 ng/ml). Peripheral blood monocytes were purified by adherence to plastic Petri dishes and treated with cold PBS supplemented with EDTA. Monocytes were cultured as previously described and stimulated with LPS (50 ug/ml). Cell-free supernatants were obtained by centrifugation and stored at –20°C, until the BGP assay was performed. The authors did not observe secretion of detectable amounts of BGP in the supernatants of short-term lymphocyte or monocyte cultures. These data indicate that circulating mononuclear cells are not involved in BGP synthesis and secretion.     

Separation and analysis of subcellular organelles in a human promyelocytic leukemia cell line, HL-60: application to the study of myeloid lysosomal enzyme synthesis and processing

We describe a system for analysis of the intracellular pathways in the biosynthesis and packaging of functionally important proteins in human myeloid cells. The human promyelocytic cell line HL-60 was used since peripheral blood neutrophils are terminally differentiated and do not actively synthesize protein. Cells were disrupted by nitrogen cavitation and subcellular organelles in postnuclear supernatant separated on a discontinuous gradient of Percoll modified to resolve organelles important in protein synthesis. This Percoll gradient separated azurophilic granules from less dense organelles and partially separated the less dense organelles from one another. Approximate densities of organelles identified by electron microscopy and by biochemical markers are azurophilic granules, 1.102 g/mL; endoplasmic reticulum, 1.039 g/mL; Golgi apparatus, 1.032 g/mL; and plasma membrane, 1.027 g/mL. We validated the utility of this method of subcellular fractionation by examining intracellular transport of myeloperoxidase, a myeloid lysosomal enzyme present in azurophilic granules. The subunits of mature myeloperoxidase (molecular weight [mol wt] = 59,000 and 13,500) cosediment with biochemical markers for lysosomes, whereas the large-mol wt (89,000) precursor forms cosediments with biochemical markers of less dense organelles. Within the limits of assay sensitivity, the 89,000-mol wt precursor is enzymatically inactive and has no spectral evidence for a heme group, suggesting that precursors of myeloperoxidase may undergo proteolytic maturation in a prelysosomal compartment with concomitant incorporation of a heme group and acquisition of enzymatic activity. This system of analysis should be suitable for the identification, subcellular localization, and maturational analysis of other myeloid lysosomal enzymes as well as functionally important membrane proteins.     

Biosynthesis of calcitonin by human lung cancer cells

The ectopic secretion of calcitonin (CT) by a wide variety of nonthyroidal human tumors has been studied by CT RIA, but little information is available concerning the biosynthesis of CT in these tumors. In the present study, a human lung cancer cell line (BEN), secreting high mol wt forms of CT was investigated to characterize the CT gene products synthesized. When conditioned medium from BEN cells was chromatographed through a Bio-Gel P-30 column, larger species of immunoreactive CT were detected with mol wt of approximately 8,000 and 18,000. Little, if any, CT of 3,500 mol wt was detected. To examine CT gene products produced in BEN cells, poly A+ RNA was isolated from BEN cells and subjected to cell-free translation assays and DNA/RNA hybridization assays. In the wheat germ cell-free translation assay, a single BEN cell product which migrated on sodium dodecyl sulfate-polyacrylamide gels with an apparent mol wt of 17,000 could be specifically immunoprecipitated with CT antisera. A similar sized CT-related translation product is produced in wheat germ assays programmed by mRNA prepared from human medullary thyroid carcinomas. In DNA/RNA hybridization assays, a single BEN cell mRNA species of 1,000 base pairs, identical in size to human thyroidal CT mRNA, hybridized to a radiolabeled CT cDNA probe. Hybridization of the CT cDNA probe with BEN cell mRNA was confirmed by RNA dot blot hybridization and cytoplasmic RNA blotting procedures. These results indicate that larger mol wt forms of CT secreted by BEN cells are derived from a translation product and a mRNA which are of similar, if not identical, size as CT gene products produced in human thyroidal tissues. The inability of lung tumor cells to process the CT precursor to calcitonin of 3,500 mol wt may reflect a lack of specific prohormone processing enzymes in these tumor cells or may be due to structural polymorphism in the CT precursor expressed in the lung cells.     

Nuclear thyroid hormone receptors in cultured bone cells

Thyroid hormones influence bone metabolism, but a direct interaction of triiodothyronine with nuclear T3 receptors in bone cells has not yet been reported. We investigated 125I-T3 binding to nuclei isolated from the cloned osteoblastlike rat osteosarcoma cells ROS 17/2.8. At 37 degrees C, saturable 125I-T3 binding to isolated nuclei reached equilibrium by 30 minutes and was completely displaced upon the addition of 500 nmol/L unlabeled T3. Nonsaturable binding represented about 0.5% of the radioactivity added (20% of the total binding). Thyroxine and 3,3',5'triiodothyronine competed with 125I-T3 with a 20-fold and 400-fold lower affinity than T3, respectively. Analysis of equilibrium competition experiments revealed the presence of a single class of homogeneous binding sites with an association constant of 5.0 +/- 0.3 X 10(9) mol/L-1 and a maximum nuclear binding capacity of 0.13 +/- 0.02 ng/mg DNA. A twofold increase of bone Gla protein (BGP) secretion was observed with T3 treatment suggesting that these T3 nuclear receptors are coupled with a biological response.     

Regenerating bone marrow produces a potent growth-promoting activity to osteogenic cells

It is well documented that injury to bone marrow is followed by an osteogenic phase that precedes the complete tissue regeneration. We have recently shown that postablation healing of bone marrow in rat tibiae is associated with a systemic increase in osteogenesis. It was hypothesized that a growth factor(s) with an effect on osteogenic cells is produced in the healing limb, is transferred to the blood circulation, and enhances osteogenesis systemically. To test growth factor production, healing bone marrow-conditioned medium was prepared with tissue separated from rat tibias during the osteogenic phase and assayed for enhancement of mitogenic activity in culture of osteogenic rat osteosarcoma cells (ROS 17/2). Partial purification of healing bone marrow-conditioned medium-derived growth factor(s) consisted of gel filtration on Sephadex G-25, boiling, chromatography on heparin-Sepharose, and gel filtration on Sephadex G-75. Mitogenic activity eluted in the void volume of the Sephadex G-25 column (mol wt greater than 5,000). Potent activity resolved from heparin-Sepharose with PBS, and on filtration by Sephadex G-75 this activity recovered in 3 peaks with mol wt estimates of 35,000, 19,000, and less than 10,000. The partially purified factor also showed considerable stimulatory effect on DNA synthesis in osteoblastic fetal rat calvarial cells and on in vitro elongation of fetal long bone; it had only a small effect on nonosteoblastic ROS and fetal rat calvarial cells. These data indicate that healing bone marrow produces growth factor activity with a preferential effect on osteogenic cells. It is suggested that local growth factors have a role as mediators in the sequence of events whereby bone marrow expresses its osteogenic potential. During postablation healing of bone marrow these factors may also function as systemic promoters to osteogenic cells.     

Platelet-derived growth factor enhances deoxyribonucleic acid and collagen synthesis in osteoblast-enriched cultures from fetal rat parietal bone

Platelet-derived growth factor (PDGF) or closely related proteins are found in bone matrix and are produced by cultured osteosarcoma cells. In serum-deprived osteoblast-enriched (ob) cultures from fetal rat bone, recombinant human PDGF (composed of a B chain homodimer) at 0.1-3 nM enhanced the rate of DNA synthesis by 2- to 8-fold within 24 h of treatment, and 0.3-3 nM PDGF increased cell number by 1.3- to 1.6-fold. Unlike results with rat kidney fibroblast cultures, the mitogenic effect of PDGF in ob cultures was not synergistic with that of insulin-like growth factor I. PDGF at 0.3-10 nM also enhanced the rates of collagen and noncollagen protein synthesis in ob cultures by 1.5- to 4.0-fold, and these increases were blocked when DNA synthesis was prevented. The stimulatory effects of PDGF did not appear specific to ob cultures from fetal rat bone, since similar increases were found in bone cell cultures containing fibroblasts and osteoblast precursors. PDGF binding at 4 C to ob cultures indicated a single class of receptors with a Kd of 0.16 nM and approximately 60,000 sites/cell. Polyacrylamide gel of 125I-PDGF bound and cross-linked to ob cultures revealed a single radioactive band at approximately 180,000-190,000 mol wt. The present studies, therefore, indicate that PDGF can directly increase replication and matrix protein synthesis by both differentiated and undifferentiated bone cells, and that bone- or platelet-derived PDGF may have an important anabolic role in bone remodeling or fracture repair.     

不同剂量重组人骨形态发生蛋白2对人成骨肉瘤细胞系SaOS-2成骨活性的影响

目的 探讨不同剂量的重组人骨形态发生蛋白2(rhBMP-2)对人成骨肉瘤细胞系SaOS-2成骨活性的影响.方法 将体外培养的SaOS-2细胞分别暴露于0(对照)、2、20、200μg/L的rhBMP-2中,培养12、24、48 h后,以实时荧光定量PCR方法测定SaOS-2细胞碱性磷酸酶(ALP)和骨钙素(BGP)mRNA的表达水平.结果 SaOS-2细胞ALP和BGP mRNA表达量随rhBMP-2刺激剂量的增加呈上升趋势.20μg/L rhBMP-2作用48 h(1.60±0.64)和200 μg/L rhBMP-2作用12、48 h(1.70±0.41、1.80 ±0.19)SaOS-2细胞ALP mRNA 相对表达量较同一时间段对照组(12、48 h分别为0.80±0.25、0.74±0.21)明显升高(P均<0.05).2μg/LrhBMP-2作用24 h(1.67±0.33)、20μg/L rhBMP-2作用12、24 h(2.42±0.13、1.82±0.14)和200 μg/L rhBMP-2作用12、24 h(1.46±0.11、1.24 ±0.07)SaOS-2细胞BGP mRNA相对表达量较同一时间段对照组(12、24 h分别为1.01±0.14、0.84±0.12)明显升高(P均<0.05).结论 rhBMP-2能明显刺激SaOS-2细胞ALP、BGP mRNA表达升高,并呈现剂量一效应关系,但表现的剂量特征不同.

Abstract：

Objective To investigate the effect of recombinant human bone morphogenetic protein 2 (rhBMP-2) on the osteogenic activities of human osteosarcoma cell line SaOS-2. Methods SaOS-2 cells were exposed to rhBMP-2 for 12,24,48 h at 0(control) ,2,20,200 μg/L, respectively. The mRNA expression of alkaline phosphatase(ALP) and bone gla(BCP) were detected by real time polymerase chain reaction. Results The mRNA expression of ALP and BGP of SaOS-2 cells increased gradually with rhBMP-2. The mRNA expression of ALP of the 20 μg/L group exposed for 48 h(1.60 ± 0.64), and the 200 μg/L group exposed for 12,48 h(1.70 ± 0.41, 1.80±0.19) were significantly higher than those of control (12 h: 0.80±0.25, 48 h: 0.74±0.21, allP<0.05). The mRNA expression of BGP of the 2 μg/L group exposed for 24 h(1.67 ± 0.33), the 20 μg/L group exposed for 12,24 h(2.42 ± 0.13,1.82 ± 0.14) and the 200 μg/L group exposed for 12,24 h(1.46 ± 0.11,1.24 ± 0.07) were significantly higher than those of control( 12 h: 1.01 ± 0.14, 24 h: 0.84 ± 0.12, all P< 0.05). Conclusions rhBMP-2 can promote the mRNA expression of ALP and BGP of SaOS-2 cells. They have a dose-response relationship, but represent a different dose-response effect.     

Interferon-gamma inhibits 1,25-dihydroxyvitamin D3-stimulated synthesis of bone GLA protein in rat osteosarcoma cells by a pretranslational mechanism

Interferon-gamma (IFN) is produced by lymphocytes in areas of inflammation and connective tissue destruction. IFN inhibits collagen and DNA synthesis in cultured rat long bones and osteoblastic ROS 17/2.8 cells, suggesting that the periarticular loss of bone that occurs in inflammatory joint diseases may be due to IFN inhibition of bone formation. Since serum levels of bone gla protein (BGP) have been correlated with the bone formation rate, we studied the effect of IFN on production of this osteoblast-specific protein and steady state BGP messenger RNA (mRNA) levels in ROS 17/2.8 cells. RIA of BGP was done using an antibody raised against rat BGP peptide. BGP synthesis was stimulated with 10(-8) M 1,25-dihydroxyvitamin D3 24 h before and continuously after addition of recombinant rat IFN. IFN (100 U/ml) inhibited BGP secretion 52%, 78%, and 70% in the first, second, and third 24 h periods after IFN treatment, compared to control cells cultured with 1,25-dihydroxyvitamin D3 alone. The ED50 for IFN inhibition of BGP production was 3.3 U/ml (0.29 nM). Pulse labeling with [14C]leucine or [3H]proline during the last 4 h of culture revealed that IFN (3-100 U/ml) did not inhibit total protein secretion into the medium. The percent inhibition of BGP production by IFN was independent of media serum concentration or cell density. IFN (100 U/ml) decreased the steady state level of BGP mRNA as measured by Northern analysis using an oligomeric probe for rat BGP. The decrease in hybridization signal for BGP mRNA was detectable by 1 h after IFN exposure and continued to decline at 6 and 24 h. Treatment with cycloheximide (5 micrograms/ml) blocked the inhibitory effect of IFN on steady state levels of BGP mRNA. These results suggest that IFN may inhibit bone formation by selective inhibition of osteoblast matrix protein production. The mechanism of IFN inhibition of BGP production is, at least in part, pretranslational.     

A radioimmunoassay for bone Gla protein (BGP) in human plasma

To study the value of bone Gla protein (BGP) as a biochemical marker of normal bone physiology and metabolic bone disorders, we have developed a radioimmunoassay (RIA) for the detection of BGP in human plasma. Antibodies were generated in rabbits immunized with purified calf BGP conjugated to thyroglobulin. Human plasma BGP reacted identically with the calf BGP standard, thus demonstrating the suitability of the assay to measure plasma BGP levels in man. The RIA is sensitive, accurate, and technically simple. Plasma BGP levels were determined in normal subjects (N = 35) and in patients with hypothyroidism (N = 10), hyperthyroidism (N = 22) and chronic renal failure (N = 35). The mean (+/- 1 SEM) concentration of plasma BGP in normal subjects was 1.27 +/- 0.07 nmol/l. Plasma BGP was significantly increased in patients with hyperthyroidism, 4.04 +/- 0.78 nmol/l (P less than 0.001) and chronic renal failure, 10.17 +/- 2.47 nmol/l (P less than 0.001). Low concentrations were found in patients with hypothyroidism, 0.74 +/- 0.11 nmol/l (P less than 0.01). Our studies indicate that plasma BGP provides a useful technique in the diagnosis of patients with bone disease.     

Precursors of alpha-inhibin modulate follicle-stimulating hormone receptor binding and biological activity

Although several forms of monomeric alpha-inhibin have been isolated from follicular fluid, no biological function has yet been ascribed to these posttranslationally processed forms of the alpha-subunit precursor protein. Moreover, previous studies of a FSH receptor binding competitor (FRBC) isolated and characterized from porcine follicular fluid (pFF) suggested certain biochemical similarities between this protein and alpha-inhibin precursors. We, therefore, investigated the hypothesis that alpha-inhibin and/or its precursors might represent autocrine and/or paracrine modulators of FSH action in the ovary, accounting for some of this FRBC activity and thereby exerting some degree of regulation over follicular maturation. Three separate sources of alpha-inhibin proteins were investigated for FRBC activity, including pFF, human FF (hFF), and a 293 cell line into which the full-length human alpha-inhibin cDNA had been stably transfected. Conditioned medium from these transfected cells contained several forms of alpha-inhibin precursors as well as mature alpha-inhibin, but no beta-subunit or intact inhibin. alpha-Inhibin proteins from all three sources, purified by a variety of methods, including immunoaffinity chromatography on an anti-alpha-inhibin column, inhibited FSH binding to both natural tissue FSH receptors as well as recombinant rat FSH receptors expressed in 293 cells. Furthermore, dimeric inhibin and activin, medium from untransfected 293 cells, and non-alpha-inhibin-containing purification fractions were inactive in either assay. In addition, purified recombinant alpha-inhibin proteins were partial in vitro FSH antagonists in a bioassay in which cAMP generation from 293 cells expressing the recombinant FSH receptor is used as an index of FSH biological activity. These same fractions of hFF containing FRBC activity did not bind to LH receptors, thereby demonstrating receptor specificity for this activity. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with alpha-inhibin or FRBC antisera, a 57,000 mol wt protein was identified in FRBC-active fractions from all three sources, suggesting that the active moiety was the full-length alpha-inhibin precursor protein or a large mol wt fragment, but not mature alpha-inhibin. Lastly, all FRBC activity from all three sources was extracted by an alpha-inhibin immunoaffinity column and was recoverable upon elution. These results demonstrate that proteins derived from the alpha-inhibin precursor modulate FSH binding to its receptor as well as its biological activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Antibody against acid-stable insulin-like growth factor binding protein detects 150,000 mol wt growth hormone-dependent complex in human plasma

In order to measure the 150,000 mol wt binding complex for insulin-like growth factors in human plasma without the need for acidification to dissociate endogenous ligands, we have purified an acid-stable plasma binding protein (BP) and raised a specific rabbit antiserum against it. When used in RIA, with a covalent complex of binding protein and radioiodinated insulin-like growth factor-I as tracer, pure BP and human plasma samples gave parallel displacement curves. Incubation of plasma with pure insulin-like growth factor-I or -II (5 micrograms/ml) had no effect on the immunoreactivity of the samples. Compared to purified BP standard, the immunoreactive BP content of plasma from normal adults was 9.20 +/- 1.59 micrograms/ml (mean +/- SD, n = 10), from acromegalic subjects, 22.5 +/- 3.63 micrograms/ml (n = 10), and from GH-deficient subjects, 4.10 +/- 1.59 micrograms/ml (n = 7). Although this antibody reacted with a protein of approximately 60,000 mol wt in acidified plasma, gel chromatography of plasma at pH 7 indicated that a GH-dependent protein of mol wt 150,000 was measured almost exclusively in unacidified samples. Direct binding of insulin-like growth factor-II, however, showed the major peak of binding activity in normal plasma in the 40-45,000 mol wt region, whereas in acromegalic plasma most ligand binding was in the 150,000 mol wt region. It is concluded that there is essentially no free immunoreactive acid-stable BP in native plasma, that the unoccupied binding sites of 40-45,000 mol wt, detectable by ligand binding, are present on an immunologically distinct protein, and that the immunoreactive 150,000 mol wt binding complex in native plasma is strongly GH-dependent.     

Production of B cell growth factor(s) by neoplastic B cells from hairy cell leukemia patients

Recent studies have shown that normal human T cells contain a high- molecular-weight (mol wt) protein exhibiting B cell growth factor (BCGF) activity. Other studies have shown that virally transformed human B cells also secrete a high-mol-wt BCGF-like molecule in vitro. We have studied neoplastic B cells from patients with untreated hairy cell leukemia (HCL) to ascertain whether such cytoplasmic BCGF activity is present in the tumor cells. Studies on HCL cells from four patients indicated that BCGF-like activity was in fact present in the cytosolic extracts when tested on autochthonous HCL cells as well as on normal BCGF-dependent human B cell lines. Chromatographic analysis indicated that the BCGF activity from HCL cells was similar in mol wt as well as function to the normal T cell-derived cytosolic BCGF activity. These studies suggest that HCL cells contain and, in some cases, secrete a high-mol-wt growth factor that can be autostimulatory and appears to resemble a similar growth factor molecule found in normal human T cells.     

Isolation of an inhibitory insulin-like growth factor (IGF) binding protein from bone cell-conditioned medium: a potential local regulator of IGF action.

Inhibitory insulin-like growth factor binding protein (In-IGF-BP) has been purified to homogeneity from medium conditioned by TE89 human osteosarcoma cells by two different methods using Sephadex G-100 gel filtration, FPLC Mono Q ion-exchange, HPLC C4 reverse-phase, HPLC CN reverse-phase, and affinity chromatographies. In-IGF-BP thus purified appeared to be homogeneous and unique by the following criteria. (i) N-terminal sequence analysis yielded a unique sequence (Asp-Glu-Ala-Ile-His-Cys-Pro-Pro-Glu-Ser-Glu-Ala-Lys-Leu-Ala). (ii) Amino acid composition of In-IGF-BP revealed marked differences with the amino acid compositions of other known BPs. (iii) In-IGF-BP exhibited a single band with a molecular mass of 25 kDa under reducing conditions on sodium dodecyl sulfate/polyacrylamide gels. IGF-I and IGF-II but not insulin displaced the binding of 125I-labeled IGF-I or 125I-labeled IGF-II binding to In-IGF-BP. In-IGF-BP inhibited basal, IGF-stimulated bone cell proliferation and serum-stimulated bone cell proliferation. Forskolin increased synthesis of In-IGF-BP in TE85 human osteosarcoma cells in a dose-dependent manner. Based on these findings, we conclude that In-IGF-BP is a protein that has a unique sequence and significant biological actions on bone cells.     

Synthesis of a gene encoding parathyroid hormone-like protein-(1-141): purification and biological characterization of the expressed protein

PTH-like proteins (PTHLP), which are associated with humoral hypercalcemia of malignancy, have recently been purified. Isolation of their corresponding cDNAs has revealed that they are derived from a single gene. In this report a synthetic gene encoding PTHLP-(1-141), a 141-amino acid protein corresponding to the most abundant PTHLP cDNA detected in human tumors, was expressed in bacteria and purified to homogeneity. Recombinant (r) PTHLP-(1-141) migrates with an aberrantly high mol wt on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, presumably as a result of its unusually basic pI. rPTHLP-(1-141), like PTH, induced hypercalcemia in rats, caused release of 45Ca from fetal rat bones, and stimulated the synthesis of cAMP by rat osteosarcoma cells and canine renal membrane preparations. A comparison of the abilities of rPTHLP-(1-141) and bovine PTH-(1-34) to stimulate cAMP synthesis indicated rPTHLP-(1-141) to be 5-fold more potent in the osteosarcoma assay, while nearly 30-fold less active in the renal membrane adenylate cyclase assay. Although 100-fold less potent than bovine PTH-(1-34) in promoting bone resorption, rPTHLP-(1-141) was a potent calcemic factor in vivo, inducing a rise in serum calcium from 10.4 to 14.5 mg/dl when infused into rats at 1.3 micrograms/h. These results support previous assumptions that PTHLP is the humoral factor responsible for humoral hypercalcemia of malignancy. In addition, they suggest substantial differences between PTHLP and PTH in the regulation of calcium homeostasis.