Transforming growth factor--alpha stimulates chemotaxis of osteoblasts and osteoblast-like cells in vitro

Homeostatic turnover of bone is believed to be regulated in part by growth factors present in the surrounding environment. The first step during the bone formation process is the recruitment of osteoblasts from the surrounding intact bone to the resorption site. In this study, the effects of TGF-alpha on osteoblast chemotaxis was investigated. Cultures of rat osteoblasts and human osteoblast-like cells were examined for chemotaxis in response to increasing concentrations of TGF-alpha utilizing a modified Boyden Chamber technique. TGF-alpha stimulated a dose-dependent increase in chemotaxis by both cell populations. These results indicate that TGF-alpha may be playing an important role in the early stages of bone matrix regeneration by stimulating osteoblast chemotaxis.     

Concentration of insulin-like growth factor (IGF)-I and -II in iliac crest bone matrix from pre- and postmenopausal women: relationship to age, menopause, bone turnover, bone volume, and circulating IGFs

Insulin-like growth factor-I (IGF-I) and -II are important local regulators of bone metabolism, but their role as determinants of human bone mass is still unclear. In the present study, we analyzed the concentration of IGF-I and -II in the bone matrix of 533 human biopsies from the iliac crest that were obtained during surgery for early breast cancer. There was an inverse association of bone matrix IGF-I concentration with age that was unaffected by menopause. Bone matrix IGF-I was positively associated with histomorphometric and biochemical parameters of bone formation and bone resorption and with cancellous bone volume. Based on the estimates of the linear regression analysis, women with a bone matrix IGF-I concentration 2 SD above the mean had a 20% higher bone volume than women with a bone matrix IGF-I concentration 2 SD below the mean. In contrast, serum IGF-I was neither correlated with bone turnover nor with bone volume and was only weakly associated with bone matrix IGF-I when adjusted for the serum concentration of IGF binding protein-3. Bone matrix IGF-II was positively associated with the osteoblast surface, but in contrast to IGF-I, tended to be positively associated with age and was unrelated to cancellous bone volume. In summary, our study suggests the following. 1) The concentration of IGF-I in cancellous bone undergoes age-related decreases that are similar to those of circulating IGF-I. 2) Menopause has no effect on this age-related decline. 3) Physiological differences in bone matrix IGF-I are associated with differences in iliac crest cancellous bone volume. 4) Bone matrix IGF-I is a better predictor of cancellous bone volume than circulating IGF-I. 5) The role of IGF-II in human bone tissue is clearly distinct from that of IGF-I.     

Recombinant human bone morphogenetic protein-2 stimulates osteoblast differentiation and suppresses matrix metalloproteinase-1 production in human bone cells isolated from mandibulae

Bone morphogenetic protein (BMP), a member of the transforming growth factor superfamily, is one of the most potent growth factors that stimulate osteoblast differentiation and bone formation. We investigated the effects of recombinant human BMP-2 (rhBMP-2) on osteoblast differentiation and matrix metalloproteinase-1 (MMP-1) production in human bone cells (HBC) isolated from mandibulae of 3 adult patients. rhBMP-2 at concentrations over 50 ng/ml significantly stimulated alkaline phosphatase activity and parathyroid hormone (PTH)-dependent 3', 5'-cyclic adenosine monophosphate accumulation, which are early markers of osteoblast differentiation, in HBCs. rhBMP-2 (500 ng/ml) also enhanced the level of PTH/PTH related-peptide receptor mRNA expression in HBCs. Although neither HBCs untreated nor treated with rhBMP-2 produced measurable amounts of osteocalcin, which is a marker of more mature osteoblasts, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] induced ostocalcin mRNA expression and its protein synthesis in these cells. rhBMP-2 inhibited 1,25(OH)2D3-induced osteocalcin synthesis in HBCs at both the mRNA and protein level. rhBMP-2 also significantly suppressed MMP-1 production and MMP-1 mRNA expression at concentrations over 500 ng/ml. These results suggest that rhBMP-2 exerts anabolic effects on human osteoblastic cells derived from mandibulae by stimulation of osteoblast differentiation and down-regulation of MMP-1 synthesis.     

Effects of insulin-like growth factor I and II on DNA synthesis and proliferation in primary cultures of adult rat hepatocytes

We compared the effects of insulin-like growth factor I (IGF-I) and II (IGF-II) on DNA synthesis and proliferation and investigated various signal transduction mechanisms involved in insulin-like growth factor-induced mitogenesis in primary cultures of adult rat hepatocytes. IGF-I stimulated hepatocyte DNA synthesis and proliferation with an EC50 of 75 ng/ml within 4 h of culture. These effects were sensitive to the IGF-I concentration and cell density. Hepatocyte proliferation induced by IGF-I was potentiated by metaproterenol (10(-6) M) as well as by 8-bromo-cAMP, phorbol 12-myristate 13-acetate (PMA; 10(-8) M) and was inhibited by U-73122 (1-(-[[17beta-3-methoxyestra-1,3,5(10)-triene-17-yl]amino]hexyl]-+ ++1Hpyrrol-2,5-dione)), genistein, wortmannin, PD98059 (2'-amino-3'-methoxyflavone) and rapamycin. The IGF-I effect was independent of pertussis toxin (100 ng/ml). IGF-II also dose dependently stimulated hepatocyte DNA synthesis and proliferation with an EC50 of 0.75 ng/ml within 4 h of culture. However, these effects were not dependent on the initial plating density. The stimulatory effects of IGF-II were potentiated by UK-14304 (5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline) (10(-5) M) and inhibited by phenylephrine, PMA, metaproterenol, 8-bromo-cAMP, PD98059, rapamycin, and pertussis toxin. The IGF-II effects were not affected by genistein, U-73122, and wortmannin. These results suggest that IGF-I and IGF-II rapidly stimulate the DNA synthesis and proliferation of adult rat hepatocytes by separate mechanisms.     

Tissue distribution of DNA adducts in rats treated by intramammillary injection with dibenzo[a,l]pyrene, 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene

Recombinant human bone morphogenetic protein (rhBMP-2) was examined for its in vitro effects on biochemical markers representing osteoblast phenotype. Primary cultures of fetal rat calvarial osteoblasts were used in this study. The results indicated that rhBMP-2 stimulated alkaline phosphatase activity, parathyroid hormone (PTH)-induced cyclic AMP production, and collagen biosynthesis in a dose-dependent manner in confluent cultures. The percent collagen synthesis also increased in a dose-dependent manner. Alkaline phosphatase activity was stimulated in a time-dependent manner by rhBMP-2 that reached its maximum 5 days after initiation. Cycloheximide (2 micrograms/ml) inhibited rhBMP-2-stimulated alkaline phosphatase indicating de novo protein synthesis of the enzyme. Transforming growth factor-beta 1 (TGF-beta 1)-induced inhibition of alkaline phosphatase activity observed in confluent primary cultures was completely abolished by rhBMP-2 at a concentration that was 43 times greater than the TGF-beta 1 concentration. Also, rhBMP-2 produced a small stimulation of alkaline phosphatase activity in cells grown in the absence of ascorbic acid; however, the effect was greatly enhanced in cells cultivated in the presence of ascorbic acid (50 micrograms/ml). In view of the potentiating effect of ascorbic acid on rhBMP-2-induced stimulation of alkaline phosphatase, we speculate that ascorbic acid could amplify the osteoinductive effects of rhBMP-2 and thereby augment the efficacy of the BMP when used as bone repair material in vivo. rhBMP-2 (4.3-86 ng/ml) did not exhibit mitogenic effects on cultured osteoblasts. These data suggest that rhBMP-2 has the ability to induce expression of various markers associated with the osteoblast phenotype in primary cultures of fetal rat calvarial osteoblasts. In addition, we speculate that TGF-beta 1 may play a regulatory role in BMP-induced bone formation and ascorbic acid may potentiate the effects of rhBMP-2 in vivo.     

Skeletal cell stresses and bone adaptation

BACKGROUND: We have previously shown that growth hormone (GH) consistently stimulates proliferation of human osteoblasts in vitro. In rat osteoblasts, GH augments the effects of insulin-like growth factor (IGF) I on cell proliferation and differentiation. We therefore investigated the effects of IGF-I and -II alone and in combination with GH on human osteoblasts in vitro. METHODS: Human osteoblast-like cells (HOB) were established from trabecular explants (n = 18) and human marrow stromal cells (HMS) from marrow aspiration (n = 21). The cell cultures were stimulated with IGF-I or IGF-II (1, 10 or 100 ng mL-1) alone, in combination with hGH (100 ng mL-1) or after prestimulation with hGH. RESULTS: IGF-I alone, in combination with hGH and after pretreatment with hGH, increased proliferation of HOB and HMS by 49-190% (P < 0.05-0.01). IGF-II alone, in combination with hGH and after pretreatment with hGH increased proliferation of HOB by 57-158% (P < 0.01). In HMS only IGF-II in combination with hGH and after prestimulation with hGH increased proliferation. IGF-I alone and in combination with hGH decreased alkaline phosphatase (AP) in both cell types. IGF-II did not affect AP in HOB, but increased AP in HMS, this effect was abolished by hGH. In HOB, collagen production (PICP) was increased by IGF-II but unaffected by IGF-I. In HMS, PICP was decreased by IGF-I and -II but increased by hGH. Co-stimulation further increased PICP. CONCLUSION: IGF-I and -II exerted proliferative effects on both HOB and HMS. Co-stimulation with GH exhibited synergism in enhancing the proliferative response. In HMS prestimulation improved the proliferative response significantly. The effects of the IGFs on differentiation are more complex and dependent on cell maturation and of the IGF used.     

Insulin-like growth factor binding proteins-2 and -3 stimulate growth hormone receptor binding and mitogenesis in rat osteosarcoma cells

GH exerts its biological actions on osteoblasts through a specific high affinity receptor expressed on these cells. GH receptor binding is positively modulated by a number of factors, including retinoic acid and dexamethasone, whereas fetal calf serum strongly decreases the binding. To identify responsible factors in serum, components of serum, the insulin-like growth factors (IGFs)-I and -II, and IGF binding proteins (IGFBPs)-2 and -3 were tested for a possible negative modulatory role. IGF-I and -II decreased [125I]hGH binding at an optimal concentration of 30 ng/ml for IGF-I and 100 ng/ml IGF-II, reducing the binding to 51% and 55%, respectively, of control values. A stimulation of [125I]hGH binding was observed with IGFBP-2 as well as IGFBP-3, inducing an increase to 148% and 151% of control binding at an optimal concentration of 3000 ng/ml for both peptides. The effects of all peptides were dependent on the incubation time, being significantly increased after 8 h of incubation and reaching the full effect thereafter. The effects were declined at 24 h compared with 16 h for IGFBP-2 and -3 but not for IGF-I and -II. Coincubation of the cells with IGF-I and -II and IGFBP-2 and -3 neutralized the effects of the factors alone. In conclusion, these results show that IGF-I and -II on the one hand and IGFBP-2 and -3 on the other hand exert opposite actions on [125I]hGH binding, IGFBP-2 and -3 exerting probably an IGF-independent effect. Further, IGF-I and -II decreased GH receptor messenger RNA (mRNA) levels, as quantified by a solution hybridization ribonuclease protection assay, from 8.65 +/- 1.78 attomoles (amol)/microgram DNA (control) to 2.4 +/- 0.68 and 2.16 +/- 0.92 amol/microgram DNA, respectively. IGFBP-2 increased GH receptor mRNA levels from 5.26 +/- 1.17 (control) to 13.19 +/- 3.48. Incubation with IGFBP-3 did not result in stimulation of GH receptor mRNA levels (8.59 +/- 2.91 amol/microgram DNA). This shows that the mechanism of regulation of the GH receptor is, except for IGFBP-3, at least in part on the mRNA level. Lastly, IGFBP-2 and IGFBP-3 are mitogenic for UMR-106.01 rat osteosarcoma cells, inducing an increase in cell number to 125% and 142% of control cell counts after 48 h of incubation with 1000 ng/ml IGFBP-2 and -3, whereas IGF-I, IGF-II and Long R3 IGF-I did not stimulate proliferation. IGFBP-2 and -3 potentiate hGH induced mitogenesis at low hGH concentrations of both factors, whereas at higher concentrations no such effect is observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Relationships of serum insulin-like growth factor II concentrations to growth, compositional, and reproductive traits of swine

Insulin-like growth factors I and II (IGF-I and -II) are peptide hormones involved in metabolic regulation of growth. The objective of this study was to determine whether IGF-II concentration was predictive of growth, compositional, and reproductive traits of pigs. Forty male and sixty female pigs, divided equally between two locations, were weighed at 3-wk intervals from birth to 21 wk and bled at 9 and 21 wk of age. At each sampling, two blood samples were collected via jugular venipuncture at an interval of at least 1 h. Serum was separated and IGF-I, IGF-II, and growth hormone (GH) concentrations were determined via RIA. Traits measured included age at puberty and first parity litter size for gilts and backfat and longissimus muscle area. Blood was collected from a random sample of 52 progeny from 13 litters at 9 wk of age and serum was assayed for IGF-II concentrations. Effects of age, sex, location, and pig within sex x location on square-root transformed IGF-II concentrations were determined by analyzing data as a splitplot. Performance traits were fitted to a model including the effects of IGF-II concentration and combinations with IGF-I concentration, sex, location, and interactions. Concentrations of IGF-II were greater at 9 than at 21 wk of age (226.7 vs 159.3 ng/mL, respectively; P < .001) but did not differ between sexes. The correlation between serum IGF-II concentrations assayed from samples collected at 9 and 21 wk was .08. The partial correlations between IGF-I and IGF-II concentrations were .33 and .14 at 9 and 21 wk, respectively. The heritability of IGF-II concentration estimated from offspring-midparent regression was .08 +/- .20. Nine-week IGF-II concentration was positively associated with increased weight from weaning to 12 wk (P < .001). However, the sum of 9-wk IGF-I and IGF-II concentrations had a greater relationship to weight and gain in the growing phase than the concentration of either hormone alone. Concentration of IGF-II at 9 or 21 wk alone did not affect backfat thickness, longissimus muscle area, percentage lean, days to 100 kg, weight at 21 wk, age at puberty, or litter size. The sum of IGF-I and IGF-II concentrations was, however, associated with increased backfat and decreased days to 100 kg.     

Insulin-like growth factor system abnormalities in hepatitis C-associated osteosclerosis. Potential insights into increasing bone mass in adults

Hepatitis C-associated osteosclerosis (HCAO) is a rare disorder characterized by a marked increase in bone mass during adult life. Despite the rarity of HCAO, understanding the mediator(s) of the skeletal disease is of great interest. The IGFs-I and -II have potent anabolic effects on bone, and alterations in the IGFs and/or IGF-binding proteins (IGFBPs) could be responsible for the increase in bone formation in this disorder. Thus, we assayed sera from seven cases of HCAO for IGF-I, IGF-II, IGF-IIE (an IGF-II precursor), and IGFBPs. The distribution of the serum IGFs and IGFBPs between their ternary ( approximately 150 kD) and binary (approximately 50 kD) complexes was also determined to assess IGF bioavailability. HCAO patients had normal serum levels of IGF-I and -II, but had markedly elevated levels of IGF-IIE. Of the IGFBPs, an increase in IGFBP-2 was unique to these patients and was not found in control hepatitis C or hepatitis B patients. IGF-I and -II in sera from patients with HCAO were carried, as in the case of sera from control subjects, bound to IGFBP-3 in the approximately 150-kD complex, which is retained in the circulation. However, IGF-IIE was predominantly in the approximately 50-kD complex in association with IGFBP-2; this complex can cross the capillary barrier and access target tissues. In vitro, we found that IGF-II enhanced by over threefold IGFBP-2 binding to extracellular matrix produced by human osteoblasts and that in an extracellular matrix-rich environment, the IGF-II/IGFBP-2 complex was as effective as IGF-II alone in stimulating human osteoblast proliferation. Thus, IGFBP-2 may facilitate the targeting of IGFs, and in particular IGF-IIE, to skeletal tissue in HCAO patients, with a subsequent stimulation by IGFs of osteoblast function. Our findings in HCAO suggest a possible means to increase bone mass in patients with osteoporosis.     

Specificity in transcriptional regulation in the absence of specific DNA binding sites: the case of T7 lysozyme

Once osteoblasts have completed their bone-forming function, they are either entrapped in bone matrix and become osteocytes or remain on the surface as lining cells. Nonetheless, 50-70% of the osteoblasts initially present at the remodeling site cannot be accounted for after enumeration of lining cells and osteocytes. We hypothesized that the missing osteoblasts die by apoptosis and that growth factors and cytokines produced in the bone microenvironment influence this process. We report that murine osteoblastic MC3T3-E1 cells underwent apoptosis following removal of serum, or addition of tumor necrosis factor (TNF), as indicated by terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling and DNA fragmentation studies. Transforming growth factor-beta and interleukin-6 (IL-6)-type cytokines had antiapoptotic effects because they were able to counteract the effect of serum starvation or TNF. In addition, anti-Fas antibody stimulated apoptosis of human osteoblastic MG-63 cells and IL-6-type cytokines prevented these changes. The induction of apoptosis in MG-63 cells was associated with an increase in the ratio of the proapoptotic protein bax to the antiapoptotic protein bcl-2, and oncostatin M prevented this change. Examination of undecalcified sections of murine cancellous bone revealed the presence of apoptotic cells, identified as osteoblasts by their proximity to osteoid seams and their juxtaposition to cuboidal osteoblasts. Assuming an osteoblast life span of 300 h and a prevalence of apoptosis of 0.6%, we calculated that the fraction that undergo this process in vivo can indeed account for the missing osteoblasts. These findings establish that osteoblasts undergo apoptosis and strongly suggest that the process can be modulated by growth factors and cytokines produced in the bone microenvironment.     

Evidence for the requirement of autocrine growth factors for development of mouse preimplantation embryos in vitro

The mitotic stimuli in the early mammalian embryo have not been unequivocally identified. One hypothesis is that the embryo releases autocrine growth factors (GFs) that have a role in such growth. To determine whether such putative GFs were limited by dilution, and hence secreted, development was observed at various embryo concentrations in culture. Embryos were collected at the zygote or 2-cell stage. Zygotes were produced by fertilization in situ (ISF) or in vitro (IVF). Two-cell-stage embryos had a high rate of development to the blastocyst stage across an embryo concentration range of 1/microl-0.001/microl. By contrast, zygotes produced by either ISF or IVF were adversely affected by reducing the embryo concentration over this range (p < 0.001), with approximately 80% of ISF zygotes developing to blastocysts at the highest concentration but only 26% at the lowest. For IVF zygotes the corresponding results were 64% and 6%. For all three embryo types, the number of cells in each blastocyst was significantly lower with reduced embryo concentration. The major determinant of zygote development was the concentration of embryos in culture rather than the absolute volume of culture medium or the actual number of embryos present. A concentration of 1 embryo/microl (in the form of 10 embryos/10microl) gave the best development rates and highest cell numbers per blastocyst. Varying the albumin concentration influenced development rates; a 10-fold reduction in BSA concentration (to 0.3 mg/ml) resulted in significantly more IVF zygotes developing to the blastocyst stage. Platelet-activating factor (PAF) is released by embryos, and albumin can act as a competitive inhibitor of PAF's action on cells. ISF embryos released more PAF (p < 0.05) into media than did similarly treated IVF embryos. There was no difference in the amount of PAF remaining associated with the resulting 2-cell embryos. The amount of PAF released by both these groups was markedly less (p < 0.001) than the amount released by 2-cell embryos collected fresh from the reproductive tract and cultured for 24 h. PAF supplementation of media caused a significant increase in the rate of blastocyst development of IVF zygotes at embryo concentrations of 0.1/microl (1 ng/ml) and 0.01/microl (100 ng/ml). Insulin-like growth factor (IGF)-I (30 ng/ml) and IGF-II (1 ng/ml) also stimulated development of IVF zygotes when cultured at an embryo concentration of 1/10 microl. Epidermal growth factor was without effect over the range 0.2-2000 ng/ml. Supplementation of media with both PAF and IGF-II gave no additional benefit over that caused by IGF-II alone, but this treatment was marginally better (p < 0.05) than PAF treatment alone. The results show that factors necessary for normal embryo development are diluted to suboptimal levels during culture at low embryo concentration. The ability of PAF, IGF-I, and IGF-II to partially compensate for the adverse effects of low embryo concentration during culture is consistent with their having roles as autocrine embryotrophic factors. The use of IVF and low embryo concentrations in culture may provide a functional multiple ablation model that will help to define the range of GFs required for normal embryo development.     

Immune responses to intestinal parasites: protection, pathology and prophylaxis

It has been suggested that adjunctive growth hormone (GH) therapy improves ovarian response and in vitro fertilization (IVF) outcome in specific groups of patients. The correlation between insulin-like growth factor (IGF) and GH is well established. The aim of this study was to determine whether changes in plasma GH correlate with IGF blood levels in patients during IVF treatment. Thirty-six women undergoing IVF and embryo transfer (ET) were examined. Ovarian stimulation was carried out by gonadotropin-releasing hormone agonists (GnRHa) and gonadotropins. Blood was drawn at the early and late follicular phase, on the day of human chorionic gonadotropin (hCG) injection and at the mid- and the late luteal phases. The samples were assayed for IGF-I, IGF-II, IGF-binding protein-3 (IGF BP-3), GH and estradiol. According to the IGF-I and GH plasma levels, patients were divided into three major groups: Group I consisted of patients in whom peak levels of GH reached more than 4 ng/ml and IGF-I decreased significantly. In this group, estradiol levels were 1863 +/- 149 pg/ml. Group II consisted of patients in whom peak blood GH levels did not exceed 2.5 ng/ml and the IGF-I level remained unchanged. In this group estradiol levels were 630 +/- 57 pg/ml. Group III consisted of patients in whom blood GH levels were low and remained unchanged while estradiol levels were 1600 +/- 420 pg/ml. In this group no significant increase in IGF-levels were observed. There was no significant change in the levels of either IGF-II or IGF BP-3 in any of the groups. We can conclude that (1) there is a negative correlation between GH and IGF-I plasma levels in patients undergoing controlled ovarian hyperstimulation (COH)-IVF, when levels of estradiol and GH are elevated; (2) plasma levels of IGF-I under ovarian hyperstimulation are probably regulated by a multifactorial system; and (3) no correlation was found between the plasma levels of IGF-I and those of IGF-II and IGF BP-3 in all patient groups.     

Effect of growth factors on growth of bovine parathyroid cells in serum-free medium

Published work has suggested a possible role of the epidermal growth factor receptor (EGFr) in parathyroid disease. Bovine parathyroid cells (BPCs) are frequently used as a tissue model for studying parathyroid disorders. We have studied the effect of the EGFr ligands EGF and transforming growth factor alpha (TGFalpha), alone and with insulin-like growth factors IGF-I and IGF-II on BPC growth. Experiments were run in triplicate and repeated three times. Cell numbers were assessed on day 5 by colorimetric MTT assay as well as by tritiated thymidine uptake. Results show that TGFalpha alone (p < 0.05) and IGF-I and IGF-II alone (p < 0.05) significantly stimulated growth over controls (t-test). Furthermore, the combination of TGFalpha with IGF-I and IGF-II exhibited significant enhancement above that seen with IGF-I and IGF-II alone (p < 0.01). EGF did not stimulate growth over controls. EGFr may be expressed in BPCs, but TGFalpha exhibits a more potent growth stimulus than EGF. Addition of IGF-I or IGF-II to the growth medium further enhances this effect.     

Random amplified polymorphic DNA (RAPD) sequences as markers for canine genetic studies

Our knowledge of the concentration of growth factors in growing bone is limited. In the present study, we examined the developmental changes in the concentrations of insulin-like growth factor I (IGF-I) and transforming growth factor beta (TGF-beta) in the rat femur between weanling and maturity. We show that during the rapid growth phase there is a continuous rise in bone matrix IGF-I and TGF-beta in all compartments of the femoral bone. The association between IGF-I and TGF-beta is not only temporal, but with few exceptions is also observed within the animals of each age class. These data support the hypothesis that IGF-I and TGF-beta play an important role in the growth-associated accumulation of bone mass.     

Growth hormone receptor activity is stimulated by insulin-like growth factor binding protein 5 in rat osteosarcoma cells

Osteoblast-like UMR-106.01 rat osteosarcoma cells express high affinity growth hormone (GH) receptors (GHRs). Because osteoblasts secrete insulin-like growth factor binding protein-5 (IGFBP-5), we evaluated whether it also modulates GH binding and GHR expression in UMR cells. Human recombinant intact IGFBP-5 stimulated 125I-hGH binding in a dose-dependent manner (dose range 300-3000 ng/ml), inducing an increase to 193.6 +/- 2.1% of control binding at 3000 ng/ml (P < 0.001). Carboxy-truncated IGFBP-5 also stimulated GH binding but with less potency (125 +/- 2.7% of control at 3000 ng/ml, P < 0.01). GHRs identified by chemical crosslinking of 125I-hGH to cell monolayers increased after treatment with IGFBP-5 and decreased in response to insulin-like growth factor-I (IGF-I). GHR mRNA levels, as quantitated by a solution hybridization RNAse protection assay, increased up to 3 to 7-fold in a time-dependent manner by intact IGFBP-5 but not by carboxy-truncated IGFBP-5. An antiserum to IGFBP-5 reduced basal GH binding to 56.7 +/- 4.3% of control value at a concentration of 0.5% (P < 0.001), showing that IGFBP-5 produced by the cells is a strong regulator of GH binding. IGFBP-5 antiserum also decreased GH binding to 85.9 +/- 0.9% of IGFBP-5 stimulated value (P < 0.001), showing the specificity of IGFBP-5 stimulation. To determine whether the GHR upregulation was physiologically significant, cell proliferation was evaluated after coincubation of IGFBP-5 with low, non-stimulatory concentrations of GH. IGFBP-5 (1000 ng/ml) induced cell proliferation to 116.2 +/- 3.2% of control levels, and coincubation with hGH at 10 ng/ml induced an increase to 133.3 +/- 0.1% of control levels. We conclude that exogenous and endogenous IGFBP-5 upregulate GHR mRNA levels and GH binding and this interaction potentiates GH-stimulated mitogenesis in osteoblastic cells.     

Localisation of insulin-like growth factor receptors in skin follicles of sheep (Ovis aries) and changes during an induced growth cycle

Pelage growth cycles are regulated by circulating prolactin in many mammals, but the intercellular mediators of this signaling are unknown. Binding sites for insulin-like growth factors (IGFs) were examined in sheep skin to show changes in distribution and abundance of IGF receptors associated with a prolactin stimulus and the subsequent hair follicle growth cycle. Follicle cycles were induced in New Zealand Wiltshire ewes by a surge in plasma prolactin following a 4-month period of prolactin suppression with bromocriptine. Eight treated and three control sheep were slaughtered at intervals over 43 days during the follicle growth cycle. At 12-20 days after the elevation of prolactin, wool follicles passed through brief catagen and telogen phases, followed by a return to anagen. IGF binding sites were localized in skin sections by incubation with 125I-IGF-I or 125I-IGF-II. Displacement with competitive binding inhibitors (unlabeled IGF-I, IGF-II, des(1-3)IGF-I, des(1-6)IGF-II, or insulin) and affinity cross-linking showed that these binding sites were predominantly IGF type 1 and type 2 (mannose-6-phosphate) receptors. The radioligands bound especially to follicle germinal cells and prekeratinocytes. Increases in specific binding of both radioligands were observed after the rise in prolactin, but prior to anatomical changes in follicles associated with cessation of growth. For IGF-I, highest binding density was observed during catagen in the germinal matrix and dermal papilla cells. For IGF-II, peak density occurred during late anagen/early catagen in the germinal matrix and during telogen in the dermal papilla. These cycle associated changes in receptor availability suggest that IGF receptors are involved in control of the wool growth.     

Transient exposure of human myoblasts to tumor necrosis factor-alpha inhibits serum and insulin-like growth factor-I stimulated protein synthesis

Tumor necrosis factor-alpha (TNF-alpha) induces cachexia and is postulated to be responsible for muscle wasting in several pathophysiological conditions. The purpose of the present study was to investigate whether exposure of human myoblasts to TNF-alpha could directly inhibit the ability of serum or insulin-like growth factor I (IGF-I) to stimulate protein synthesis as assessed by the incorporation of [3H]phenylalanine into protein. Serum and IGF-I stimulated protein synthesis dose dependently. Half-maximal stimulation of protein synthesis occurred at 05% serum and 8 ng/ml of IGF-I, respectively. TNF-alpha inhibited IGF-I-stimulated protein synthesis in a dose-dependent manner. Additionally, as little as 2 ng/ml of TNF-alpha impaired the ability of IGF-I to stimulate protein synthesis by 33% and, at a dose of 100 ng/ml, TNF-alpha completely prevented the increase in protein synthesis induced by either serum or a maximally stimulating dose of IGF-I. Inhibition of protein synthesis was independent of whether TNF-alpha and growth factors were added to cells simultaneously or if the cells were pretreated with growth factors. Exposure ofmyoblasts to TNF-alpha for 10 min completely inhibited serum-induced stimulation of protein synthesis. TNF-alpha inhibited protein synthesis up to 48 h after addition of the cytokine. TNF-alpha also inhibited serum-stimulated protein synthesis in human myoblasts that were differentiated into myotubes. In contrast, exposure of myoblasts to TNF-alpha had no effect on IGF-I binding and failed to alter the ability of either IGF-I or serum to stimulate [3H]thymidine uptake. These data indicate that transient exposure of myoblasts or myotubes to TNF-alpha inhibits protein synthesis. Thus, the anabolic actions of IGF-I on muscle protein synthesis may be impaired during catabolic conditions in which TNF-alpha is over expressed.     

Direct visualization of individual DNA molecules by fluorescence microscopy: characterization of the factors affecting signal/background and optimization of imaging conditions using YOYO

The cellular mechanisms involved in osteoblast function and bone formation alterations in osteoporosis have been partly elucidated. Recent studies have shown that bone formation abnormalities in various forms of osteopenia result mainly from defective recruitment of osteoblastic cells. These abnormalities in osteoblast function and bone formation are associated with alterations in the expression or production of several growth factors, such as IGFs and TGF-beta, which modulate the proliferation and activity of bone-forming cells. Bone loss related to aging or unloading is characterized by diminished osteoblast proliferation and reduced local concentrations of IGFs and TGF beta. In contrast, estrogen deficiency increases osteoblast proliferation and IGF-I production. These data suggest that alterations in the production of and/or in cell responsiveness to local growth factors may contribute to the bone formation abnormalities seen in these osteopenic disorders. This suggests that preventive or curative treatment with growth factors may be beneficial in osteopenia due predominantly to decreased bone formation. Low doses of IGF-I or TGF-beta have been reported to increase osteoblast recruitment and differentiation, leading to enhanced trabecular bone formation and decreased bone loss in models of osteopenia induced by aging, estrogen deficiency and unloading. A few clinical trials also suggest that low doses of growth factors may stimulate bone formation. Although these findings open up new prospects for the prevention and treatment of osteopenic disorders, progress in this direction awaits the development of factors or analogs that are capable of locally and specifically increasing osteoblast recruitment and differentiation without including side-effects.