The synthetic phytoestrogen, ipriflavone, and estrogen prevent bone loss by different mechanisms.

Ipriflavone (IP), a synthetic isoflavone has been reported to prevent bone loss in both postmenopausal women and ovariectomized (ovx) rats. The purpose of this study was to compare and contrast some of the bone protective mechanisms of IP to those of 17beta-estradiol (E(2)) in ovarian hormone deficiency. Forty-eight 95-day-old Sprague-Dawley rats were assigned to four groups: sham, ovx, ovx+IP, and ovx+E(2). The doses of IP and E(2) were 100 mg and 10 microg/kg body weight per day, respectively. Rats were fed a diet that contained 0.4% calcium, 0.3% phosphorus, and 0.195 nmol vitamin D(3)/g diet. After sacrifice, left femoral bone densities were measured and bone histomorphometry was performed on the proximal tibial metaphysis. Ipriflavone as well as E(2) treatment completely prevented the ovx-induced femoral bone density loss. However, in contrast to E(2), IP did not lower the ovx-induced rise in serum alkaline phosphatase (ALP) activity or insulin-like growth factor (IGF)-I and IGF binding protein (IGFBP)-3 concentrations. On histomorphometry analysis, the ovariectomy-induced increase (P < 0. 09) in bone formation rate (BFR) was significantly (P < 0.05) suppressed by E(2) treatment, whereas this higher BFR was maintained in IP-treated animals. These findings indicate that IP is effective in preventing the ovx-associated bone loss. The bone protective mechanisms of IP in ovarian hormone deficiency may be different from those of E(2) and may involve increased rates of bone formation.

Cognitive and neuroendocrine response to transdermal estrogen in postmenopausal women with Alzheimer's disease: results of a placebo-controlled, double-blind, pilot study.

Preliminary evidence from clinical studies indicates that treatment with estrogen augments cognitive function for women with Alzheimer's disease (AD). The neurobiology of estrogen, particularly its neuromodulatory and neuroprotective actions, provide a viable basis to support such cognition-enhancing effects. We conducted a placebo-controlled, double-blind, parallel-group design pilot clinical study to evaluate the cognitive and neuroendocrine response to estrogen administration for postmenopausal women with AD. Twelve women with probably AD of mild-moderate severity completed the study. During an eight week treatment period, six women received 0.05 mg/day dosage of 17 beta-estradiol via a skin patch and the remaining six wore a placebo skin patch. Subjects were randomized to equal distribution, and evaluated at baseline, at weeks 1, 3, 5, and 8 on treatment, and at weeks 9, 10, 11, and 13 off treatment. On each day of evaluation, cognition was assessed using a battery of neuropsychological tests, and blood samples were collected to measure plasma concentrations of estradiol and estrone. In addition, several neuroendocrine markers were measured in plasma to evaluate the relationship between estrogen-induced cognitive effects and fluctuations in the catecholaminergic and insulin-like growth factor systems. Significant effects of estrogen treatment were observed on attention (i.e. Stroop: number of self-corrections in the Interference condition, F[1,8] = 8.22, P < 0.03) and verbal memory (i.e., Buschke: delayed cued recall, F[3,30] = 4.31, P < 0.02). The salutary effects of estrogen on cognition were observed after the first week of treatment, and started to diminish when treatment was terminated. For women treated with estrogen, enhancement in verbal memory was positively correlated with plasma levels of estradiol (r = 0.96, P < 0.02) and negatively correlated with concentrations of insulin-like growth factor binding protein-3 (IGFBP-3) in plasma (r = -0.92, P < 0.03). Furthermore, a trend in the data was evident to suggest a negative relationship between plasma levels of insulin-like growth factor-1 (IGF-1) and verbal memory (r = -0.86, P = 0.06). Estrogen administration suppressed peripheral markers of the IGF system, as evidenced by a negative correlation between plasma concentration of estradiol and IGF-1 (r = -0.93, P < 0.03), and a trend for a similar relationship between plasma levels of estradiol and IGFBP-3 (r = -0.86, P = 0.06). With respect to the catecholamines assayed, norepinephrine was positively correlated with verbal memory (r = 0.95, P < 0.02) for women who were treated with estrogen. Furthermore, there was a trend to suggest a negative relationship between plasma epinephrine levels and the number of errors committed on a test of attention (r = -0.84, P = 0.07). In the placebo group, no significant effects of estrogen replacement were evident either on measures of cognition or on any of the neuroendocrine markers. The results of this study suggest that estrogen replacement may enhance cognition for postmenopausal women with AD. Furthermore, several markers of neuroendocrine activity may serve to index the magnitude of estrogen-induced facilitation on cognition. In addition, research findings from the present study will provide important information for the design of larger prospective clinical studies that are essential to definitively establish the therapeutic role of estrogen replacement for postmenopausal women with AD.

Retrovirally mediated overexpression of insulin-like growth factor binding protein 4: evidence that insulin-like growth factor is required for skeletal muscle differentiation.

Recent studies have indicated that the insulin-like growth factors (IGFs) stimulate skeletal myoblast proliferation and differentiation. However, the question of whether IGFs are required for myoblast differentiation has not been resolved. To address this issue directly, we used a retroviral vector (LBP4SN) to develop a subline of mouse C2 myoblasts (C2-BP4) that constitutively overexpress IGF binding protein-4 (IGFBP-4). A control C2 myoblast subline (C2-LNL6) was also developed by using the LNL6 control retroviral vector. C2-BP4 myoblasts expressed sixfold higher levels of IGFBP-4 protein than C2-LNL6 myoblasts. 125I-IGF-I cross linking indicated that IGFBP-4 overexpression reduced IGF access to the type-1 IGF receptor tenfold. At low plating densities, myoblast proliferation was inhibited, and myoblast differentiation was abolished in C2-BP4 cultures compared with C2-LNL6 cultures. At high plating densities in which nuclear numbers were equal in the two sets of cultures, C2-BP4 myoblast differentiation was inhibited completely. Differentiation was restored in C2-BP4 cells by treatment with high levels of exogenous IGF-I or with des(1-3)IGF-I, an analog of IGF-I with reduced affinity for IGFBPs. These findings confirm the hypothesis that positive differentiation signals from the IGFs are necessary for C2 myoblast differentiation, and they suggest that the present model of myogenic differentiation, which involves only negative external control of differentiation by mitogens, may be incomplete.

The effect on the insulin-like growth factor system in human prostate epithelial cells of immortalization and transformation by simian virus-40 T antigen.

The insulin-like growth factor (IGF) system has been demonstrated to be important for proliferation and differentiation in tissues. This system has also been demonstrated to be an important regulator of the growth of normal prostate epithelium and has been implicated in the process of transformation to human epithelial prostate cancer. This study examined the function of the various components of the IGF system in benign prostate epithelium (BPE), simian virus-40 (SV40)-T antigen-immortalized prostate epithelial cells, P69SV40-T (P69), and two sublines generated from the parental line by serial passage through athymic mice: one tumorigenic (M2182) and one metastatic (M12). IGF-II messenger ribonucleic acid (mRNA) and protein were detected in BPE cells, and each of the three P69 cell lines. IGF-II protein levels were significantly higher in medium collected from the P69, M2182, and M12 cells than in BPE. Proliferation in response to IGF was P69 > BPE > M2182 > M12. The proliferative responses in the four cell types were paralleled by an increase in c-jun. In addition, as the cells became progressively more tumorigenic, the basal level of c-jun mRNA increased. IGF-binding protein-2 (IGFBP-2), -3, -4, -5, and -6 could be detected in the primary epithelial cell medium; however, as the cells became progressively more tumorigenic, there was a decrease in IGFBP-2, -3, -5, and -6 in the medium. The type 1 IGF receptor (IGFr) also decreased as the cells became more tumorigenic. The M12 cells had 80% fewer receptors than the P69 cells and 70% fewer than M2182 cells. There was no change in the Kd for IGF between the cell lines. Based on these data it would appear that the difference in proliferation between the BPE cells and P69s may be due to an increased concentration of inhibitory IGFBPs in the P69 medium. The decrease in proliferation seen in response to IGF in M2182 and M12 cells compared to the P69s would appear at least in part to be due to a decreased IGFr number. IGFr mRNA is represented by 11.0- and 7.0-kilobase bands in the BPE and P69 cells, but only by an 11.0-kilobase band in M2182 and M12 cells. These data indicate that there are significant changes that occur in the IGF system during the process of malignant transformation of the prostate epithelium. The changes described in the P69 cell system are similar to those seen in vivo and suggest that an intact IGF system may be important in maintaining a differentiated epithelial cell.

Protein and messenger ribonucleic acid (mRNA) for the type 1 insulin-like growth factor (IGF) receptor is decreased and IGF-II mRNA is increased in human prostate carcinoma compared to benign prostate epithelium.

Insulin-like growth factors (IGFs) and the type 1 IGF receptor (IGF-R) are involved in normal growth and development of the human prostate. Changes in levels of IGF-R and IGFs have been shown for several malignancies. Immunohistochemistry and in situ hybridization were performed to compare the expression of IGF-R and IGF-II in vivo in prostate tissue containing benign epithelium, high grade prostate intraepithelial neoplasia (PIN), and adenocarcinoma. Messenger ribonucleic acid (mRNA) hybridization signals and immunoreactivity for IGF-R were localized primarily to epithelial cells, with less signal in stroma. IGF-R mRNA was significantly decreased by 42% in PIN and 35% in cancer cells compared to that in benign epithelium (P < 0.0001). IGF-R immunostaining was significantly decreased by 32% in PIN and by 42% in malignant epithelium compared to that in benign epithelium (P < 0.004). IGF-II mRNA was also localized primarily to epithelial cells. IGF-II mRNA was significantly increased by 30% in adenocarcinoma compared to that in benign epithelium (P < 0.03). Immunoreactivity for IGF-II was localized to both stroma and epithelium. Protein levels for IGF-II were not significantly increased in cancer cells compared to those in benign epithelium. The decrease in the type 1 IGF receptor and increase in IGF-II mRNA may affect prostate cancer proliferation and differentiation.

Insulin-like growth factor-binding proteins (IGFBP)-4, -5, and -6 in the benign and malignant human prostate: IGFBP-5 messenger ribonucleic acid localization differs from IGFBP-5 protein localization.

Insulin-like growth factor (IGF)-binding proteins (IGFBPs) modulate the actions of IGF. We have previously reported that IGFBP-2 messenger ribonucleic acid (mRNA) and protein are increased, and IGFBP-3 protein decreased in malignant prostate epithelium compared to benign epithelium. In this study, we examined the other IGFBPs secreted by prostate cells in vitro, namely IGFBP-4, -5, and 6. Immunoreactivity and mRNA signals for IGFBP-4 and -6 were localized to epithelial cells, with less signal in stroma. IGFBP-4 immunostaining and hybridization signal were significantly increased in prostate adenocarcinoma compared to those in benign epithelium. Immunostaining for IGFBP-5 was localized to the epithelium and stroma. IGFBP-5 immunoreactivity was significantly increased in malignant compared to benign epithelium. IGFBP-5 mRNA signal was not localized to epithelial cells; rather, the signal was over stromal cells surrounding the acinar structures. These cells are thought to be fibroblasts. We show that IGFBP-4 mRNA and protein and IGFBP-5 protein are increased in malignant epithelium compared to benign epithelium, that IGFBP-6 is present in benign and malignant epithelium, and that there is differential localization of IGFBP-5 mRNA and protein in prostate tissue. IGFBP-5 that is made by fibroblasts appears to be sequestered by epithelial cells. IGFBP-5 may, therefore, be a factor in cellular interactions between stromal and epithelial cells that are of fundamental importance for normal prostatic development and function.

Proteolysis of insulin-like growth factor-binding protein-3 in the male reproductive tract.

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) is produced by prostate epithelial and stromal cells and either enhances or inhibits the effects of IGF on prostate epithelial cells. The levels of this protein in the male reproductive tract may be determined in part by proteases, including prostate-specific antigen (PSA), produced by the prostate epithelium. In this study we examined the proteolytic activity of human seminal fluid on IGFBP-3. Seminal fluid and prostate massage fluid (PF) were examined for IGFBP-3 or its fragments by use of an IGFBP-3 RIA that detects intact IGFBP-3 as well as fragments, a two-site immunoradiometric assay (IRMA) that detects intact IGFBP-3 and the larger fragments, Western ligand blots (WLB), and immunoblots (WIB). In seminal fluid, IGFBP-3 was readily detectable by RIA, but was detected in only 50% of the samples assayed by IRMA. No detectable IGFBP-3 was observed by WLB with [125I]IGF-I as the ligand, but with IGF-II as the ligand, IGFBP-3 fragments at 16-17 kDa were noted. On WIB, the 16-kDa fragment of IGFBP-3 was most abundant, with a smaller amount of the 29-kDa fragment, but no intact IGFBP-3. These results indicated that most of the IGFBP-3 detected in seminal fluid was in small (< or = 16-kDa) fragments. When three or more seminal fluid samples collected 1 month apart were available from the same individual, the coefficient of variation was 10.0 +/- 1.26% (+/- SE) for IGFBP-3 by RIA vs. 73.3 +/- 11.2% for sperm counts in the same samples. In a group of 42 PF samples, the IGFBP-3 levels measured by either RIA or IRMA were approximately 3-fold higher than those in seminal fluid. Intact IGFBP-3 was detected by both WLB and WIB. There was a significant inverse correlation between PSA and IGFBP-3, measured by IRMA, in PF (r = -0.526; P < or = 0.004). Finally, in the PF of African-American men, PSA was significantly lower, and IGFBP-3 determined by IRMA was significantly higher compared to those in Caucasian men.

Insulin-like growth factor-binding protein-2 and -3 expression in benign human prostate epithelium, prostate intraepithelial neoplasia, and adenocarcinoma of the prostate.

Insulin-like growth factor (IGF)-binding proteins (IGFBPs) modulate the activity of IGFs. In vitro human prostate epithelial cells secrete IGFBP-2 and -3. In vivo IGFBP-2 is increased, and IGFBP-3 is decreased in the serum of patients with prostate cancer. Immunohistochemistry and in situ hybridization were performed to compare the expression of IGFBP-2 and -3 in vivo in prostate tissue containing benign epithelium, high grade prostate intraepithelial neoplasia (PIN), and adenocarcinoma. Immunoreactivity and messenger ribonucleic acid (mRNA) hybridization signals for IGFBP-2 and -3 were localized to epithelial cells. IGFBP-2 immunostaining intensity was significantly increased in PIN regions compared to that in normal epithelium and was further increased in malignant cells. IGFBP-2 mRNA was also significantly increased in PIN and cancer cells. IGFBP-3 immunoreactivity was significantly increased in PIN regions compared to normal epithelium; however, IGFBP-3 protein was significantly decreased in malignant cells. IGFBP-3 mRNA remained virtually unchanged in benign epithelium, PIN, and adenocarcinoma cells. These results demonstrate that increased IGFBP-2 protein in PIN and malignant cells is probably due to increased mRNA expression. However, levels of IGFBP-3 protein may be due to pre- and/or posttranslational mechanisms, including proteolysis. The changes in IGFBP-2 and -3 protein levels in prostatic tissue are in agreement with serum changes reported in patients with prostate cancer.

Altered growth and insulin-like growth factor-binding protein-3 production in PC3 prostate carcinoma cells stably transfected with a constitutively active androgen receptor complementary deoxyribonucleic acid.

The network of androgen-dependent growth factors regulating the growth and function of normal or neoplastic prostate epithelium is largely unknown. To facilitate studies directed at investigating this issue, androgen receptor-negative (AR-) PC3 prostate carcinoma cells were stably transfected with the expression plasmid CMV3 containing a constitutively active AR construct that is truncated at its hormone-binding domain (CMV-ARCA). The major characteristic of the resulting cell line (PC3-ARCA) was a growth rate approximately 35% slower than that of a control mock-transfected cell line (PC3-Neo). Of the several growth factors known to be present in the prostate, the current studies focused on the insulin-like growth factor (IGF) axis, specifically the IGF-binding proteins (IGFBPs), several of which are known to be abnormally produced by prostate cancer. Northern analysis showed that IGFBP-1 and -5 are not expressed by PC3-ARCA and -Neo cells. Western ligand and immunoblot analysis of medium conditioned by PC3-Neo and PC3-ARCA cells revealed that equal amounts of IGFBP-2, -4, and -6 were secreted. In contrast, IGFBP-3 was undetectable in the conditioned medium of PC3-ARCA cells, but normally produced by the AR- cell line PC3-Neo. IGFBP-3 disappearance from the conditioned medium of PC3-ARCA cells was transcriptionally regulated, as a marked decrement in IGFBP-3 messenger RNA was detected by S1 protection analysis. We investigated the responses of these cells to exogenously added IGF-I, IGF-II, or IGFBP-3. IGF-I and IGF-II stimulated the proliferation of PC3-ARCA cells, but not of PC3-Neo cells. IGFBP-3 had no effect when given alone. When IGFBP-3 was administered together with IGF-I or IGF-II, it further increased the mitogenic response observed in PC3-ARCA cells, but no effect on PC3-Neo cells was observed. In conclusion, our studies suggest that the presence of an active AR modulates the proliferation of transfected PC3 prostate cancer cells, and that this phenomenon occurs at least in part through the regulation of IGFBP-3 production.

Changes in IGF-I and -II expression and secretion during the proliferation and differentiation of normal rat osteoblasts.

IGF-I and -II have potent effects on proliferation and differentiation of osteoblasts in vitro. These cells secrete both IGFs and expression of these peptides is regulated by several of the hormones and growth factors that promote bone resorption and/or formation. However, the physiological role(s) of IGFs in the remodelling process of adult bone is still unclear. Some confusion may arise from results influenced, in part, by differences in the state of osteoblast development of in vitro cultures. Several laboratories have demonstrated that murine osteoblast cultures progress from proliferating preosteoblasts, to mature differentiated osteoblasts that form an extracellular matrix, to cultures that form a mineralized matrix. We have recently documented changes in IGF-binding protein expression and secretion in these cultures. To complement and extend this work, we have examined IGF-I expression and secretion and IGF-II expression during in vitro osteoblast development. Steady-state mRNA levels of both IGF-I and -II increased from the earliest time examined, day 5 in culture, to a maximum at day 11 and, thereafter, declined. IGF-I secreted into the medium also changed in a biphasic manner, but IGF-II could not be quantitated due to the sensitivity of our assay. Secretion of IGF-I was lowest between days 8 and 14. IGF-I secretion on day 5 was significantly greater than day 8. Similarly, IGF-1 secretion from day 17 to 26 was also greater than observed for days 8 to 14. If differentiation of the cells was inhibited, this late rise in IGF-I secretion was abolished.(ABSTRACT TRUNCATED AT 250 WORDS)

Separation of mouse crushed muscle extract into distinct mitogenic activities by heparin affinity chromatography.

Extracts from gently crushed adult mouse skeletal muscles (CMEs) contain potent myoblast mitogens, and may be used as a model system to investigate myotrophic factors released by adult muscles following injury. CME was separated into four peaks of mitogenic activity by heparin affinity chromatography. The fraction of CME that did not bind to heparin contained transferrin (Tf). Three peaks of mitogenic activity were eluted from the heparin-agarose columns at NaCl concentrations of 0.4 M, 0.9 M, and 2.0 M. A 46 kDa protein that shared antigenicity with the BB isoform of platelet-derived growth factor (PDGF-BB) was present in the 0.4 M NaCl eluant. Mitogenic activity in the 2.0 M NaCl peak eluted identically to purified basic fibroblast growth factor (bFGF), did not act additively to saturating amounts of purified bFGF, and was neutralized by anti-bFGF antibodies. The 0.9 M NaCl eluant acted additively to the combination of three known growth factors for myoblasts, bFGF, insulin-like growth factor 1, and epidermal growth factor, to stimulate C2 myoblast proliferation, suggesting this fraction contains a mitogenic activity which does not utilize (and hence compete for) receptors for the known mitogens for myoblasts. Additionally, the 0.9 M NaCl eluant did not stimulate proliferation of fibroblast-like cells derived from muscle tissue. The unbound, 0.4 M NaCl, 0.9 M NaCl, and 2.0 M NaCl eluants from the heparin-agarose column acted additively to one another to stimulate myoblast proliferation. Our data suggest that Tf, PDGF-BB-like molecules, bFGF-like activity, and an uncharacterized heparin-binding myoblast mitogen could be released after muscle injury and act to stimulate satellite cell proliferation.

Changes in insulin-like growth factor-binding protein expression and secretion during the proliferation, differentiation, and mineralization of primary cultures of rat osteoblasts.

Insulin-like growth factor-I (IGF-I) and IGF-II are secreted by the bone-forming osteoblast and have been shown to promote mitogenesis and/or differentiation of several of the cells involved in adult bone remodeling. The biological actions of the IGFs are modulated in a cell-specific manner by IGF-binding proteins (IGFBPs). All six IGFBPs are expressed by osteoblasts. Both in vitro and in vivo, osteoblasts progress through a developmental sequence from committed precursors to mature differentiated cells that form a mineralized extracellular matrix. We have examined IGFBP expression and secretion by rat calvarial cultures, a model system of osteoblast development, to correlate changes with the developmental stage. Differential expression and secretion of IGFBPs during osteoblast development were observed. Maximal IGFBP-2 and -5 messenger RNA (mRNA) expression occurred in proliferating preosteoblasts, whereas mature osteoblasts showed maximal expression of IGFBP-3, -4, and -6. Rat osteoblasts did not express IGFBP-1. Increases in IGFBP-2, -3, and -4 secretion lagged behind corresponding mRNA increases by 3-6 days. Whereas mRNA levels declined as the cultures mineralized, IGFBP secretion continued to increase. Inhibition of osteoblast proliferation, which promotes differentiation, resulted in an IGFBP secretory pattern that was consistent with that seen with mature cells. Conversely, an IGFBP secretion pattern characteristic of proliferating cells could be maintained for weeks if differentiation was inhibited. We conclude that the developmental stage of the osteoblast is an important determinant of IGFBP secretion. We propose that hormonal regulation that alters the developmental stage may secondarily affect IGFBP expression or secretion.

Insulin-like growth factor-binding protein-3 expression and secretion by cultures of human prostate epithelial cells and stromal fibroblasts.

Prostate-specific antigen (PSA) was recently shown to be an insulin-like growth factor-binding protein (IGFBP)-3 protease. However, only IGFBPs-2 and -4 have been identified in conditioned medium of prostate epithelial cells. Using cultures of human prostate epithelial and stromal fibroblastic cells, we examined conditioned medium and cell extracts for evidence of IGFBP-3 expression and secretion. Western ligand blotting of conditioned medium from epithelial or stromal cultures revealed the presence of IGFBPs in the molecular weight range 36-48 kDa, suggestive of IGFBP-3. Western immunoblots of these media confirmed the presence of IGFBP-3. Northern analyses of extracts of both stromal and epithelial cells showed a 2.5 kb band, the size of IGFBP-3 mRNA. We conclude that prostate cells express IGFBP-3 and that local proteolysis by PSA could modify this binding protein's actions in the prostate.

Effects of ovariectomy and estrogen on the serum levels of insulin-like growth factor-I and insulin-like growth factor binding protein-3.

To determine the effects of ovariectomy and 17 beta-estradiol (E2) on serum IGF-I and its binding proteins, female Sprague-Dawley rats, aged 95 days, were divided into four groups. Group 1 was sham-operated; groups 2, 3, and 4 were ovariectomized. Groups 3 and 4 received daily injections of 200 ng (low dose) and 5000 ng (high dose) E2/kg body wt./day, respectively and the others were given solvent vehicle. Ovariectomy resulted in a significant increase in serum IGF-I (P < 0.001) at 30 and 35 days post-surgery; the increase was prevented in animals that received low-dose E2 while high-dose E2 reduced serum IGF-I levels below those of the sham-operated controls (P < 0.01). Serum IGF-binding proteins (IGFBPs) were determined by IGF-ligand blot analysis, and the resulting autoradiograms quantified by laser densitometry. The intensity of the IGFBP-3 bands changed in parallel with serum IGF-I levels. Ovariectomy increased, low-dose E2 restored, and high-dose E2 reduced serum IGFBP-3 levels compared to the levels for the sham-operated controls. The intensities of binding protein bands smaller than those of IGFBP-3 appeared unchanged by the treatment regimens. A Western immunoblot analysis with IGFBP-3 antiserum confirmed the ligand-blot data. The changes in the levels of IGF-I and its binding proteins were accompanied by ovariectomy-induced increase in osteoblast and osteoclast numbers and loss of cancellous bone that were attenuated by E2 administration. We conclude that there is a possible role for IGF-I in the pathogenesis of the increased bone turnover that occurs early in ovarian hormone deficiency.

Human osteoblast-derived insulin-like growth factor (IGF) binding protein-5 stimulates osteoblast mitogenesis and potentiates IGF action.

Insulin-like growth factor (IGF)-binding proteins (IGFBPs) either inhibit or enhance IGF-stimulated cellular effects. While inhibition occurs by sequestration of IGF from cell-surface receptors, the exact mechanism of IGF-enhancement remains undefined. Human osteoblast-like bone cells in culture secrete several IGF-binding proteins, one of which we have previously identified as IGFBP-5. In this study we purified a 23-kDa IGFBP-5 from cultures of human osteoblast-like cells using ligand affinity chromatography and reversed-phase high performance liquid chromatography and tested its bioactivity in serum-free cultures of normal mouse osteoblast-like cells. Binding studies with radioiodinated IGF showed similar and relatively low affinities for IGF-I and IGF-II consistent with a carboxyl truncated IGF-binding protein. Mitogenic assays demonstrated that the binding protein, when coincubated with IGF-I or -II, enhanced mitogenesis. This enhancement was unique from other binding proteins in not requiring a preincubation period or serum co-factors. Furthermore, the osteoblast-derived IGFBP-5 stimulated mitogenesis in the absence of exogenous or endogenous IGF. Using radioiodinated IGFBP-5 we found that the binding protein could associate with the osteoblast surface, an effect which did not require IGF nor an interaction with IGF receptors. We suggest that osteoblast-derived IGFBP-5 may stimulate osteoblast mitogenesis in at least two ways, by association with IGF and by a second pathway that is independent of IGF receptor activation.

Identification of a low molecular weight inhibitor of osteoblast mitogenesis in uremic plasma.

A low molecular weight inhibitor of cartilage sulfation has been detected in the plasma of dialysis patients. Preliminary studies of this inhibitor have suggested that it may have a role in decreasing bone mass, possibly by suppressing bone cell proliferation. Since the in vitro bioassay of crude sulfation inhibitor preparations is relatively nonspecific, we investigated whether there might also be an inhibitor of osteoblast mitogenesis in uremic plasma. We fractionated plasma and plasma ultrafiltrates from dialysis patients by gel filtration chromatography and looked for inhibition of mitogenesis in cultured osteoblasts. Material from fractions with a molecular weight range of 750 to 900 inhibited osteoblast mitogenesis. The inhibitory effect, however, could be overcome with serum or insulin-like growth factor-I, suggesting that the mechanism of inhibition was not growth factor dependent. Further characterization of the inhibitor revealed that it was not a peptide or a polar lipid. We conclude that uremic plasma contains a bone cell mitogenic inhibitor which may have a role in regulating bone remodeling in adults and bone growth in children.

A novel human insulin-like growth factor binding protein secreted by osteoblast-like cells.

Insulin-like growth factor binding proteins (IGFBPs) modulate the cellular action of the insulin-like growth factors. Inhibition or enhancement of IGF effects by these cell-secreted binding proteins have been described. We have purified two IGFBPs (23 and 29 kDa) from media conditioned by U-2 human osteosarcoma cells using ligand-affinity chromatography and reversed phase HPLC. N-terminal amino acid analysis of the 23 kDa protein revealed a unique sequence with variable homology to IGFBPs 1-4. The 29 kDa IGFBP was found to be nearly identical to a recently reported IGFBP. Because the affinity purified U-2 IGFBPs enhanced IGF-I-stimulated osteoblast mitogenesis, we suggest that one or both of these binding proteins enhance IGF action in bone.

Low-molecular-weight peptides in bone extracts that stimulate osteoblast mitogenesis.

Although a majority of regulatory peptides elaborated by neuroendocrine cells are small, i.e., less than 50-60 residues, no low-molecular-weight, bone-derived mitogenic peptides have been described. We have size-fractionated extracts of neonatal mouse calvaria, a rapidly forming bone, and assayed for osteoblast proliferation. Mitogenic peptides with estimated sizes of 1,600, 1,050, and 770 daltons were detected. Their protein nature was demonstrated by the reduction in mitogenic activity following protease treatment. Fibroblast mitogenesis was not stimulated by any of the peptides. These data indicate that there are mitogenic peptides in bone smaller than any previously described locally-derived bone cell growth factor.

Ontogeny of peptidylglycine alpha-amidating monooxygenase activity in rapidly mineralizing bone from neonatal mouse.

Peptidylglycine alpha-amidating monooxygenase is hormonally, developmentally, and nutritionally regulated. In several tissues concomitant changes in enzyme activity and the level of expression of a known amidated peptide have been demonstrated. We report that neonatal mouse calvarium, a rapidly mineralizing bone, has detectable amidation enzyme activity. The level of activity varied 3-10-fold during the first 9 d of life. Production of one or more amidated peptides by bone may be coordinately regulated.

Glycosylation reduces the bioactivity of calcitonin.

The biological significance of peptide hormone glycosylation is uncertain. To examine the effect of Asn-linked glycosylation on calcitonin's bioactivity we purified glycosylated calcitonin from a transplantable rat medullary thyroid carcinoma. Glycosylated calcitonin constituted 2.3% of the total extracted immunoreactive calcitonin. The structure of this peptide differed from nonglycosylated calcitonin only by the oligosaccharide modification of asparagine 3. Affinity of glycosylated calcitonin for lentil lectin indicated that the oligosaccharide was a complex processed form. In a standard in vivo bioassay glycosylated calcitonin had a markedly reduced hypocalcemic activity compared to nonglycosylated calcitonin, an effect most likely due to the presence of the oligosaccharide.