Growth factor control of skeletal muscle differentiation: commitment to terminal differentiation occurs in G1 phase and is repressed by fibroblast growth factor.

Analysis of MM14 mouse myoblasts demonstrates that terminal differentiation is repressed by pure preparations of both acidic and basic fibroblast growth factor (FGF). Basic FGF is approximately 30-fold more potent than acidic FGF and it exhibits half maximal activity in clonal assays at 0.03 ng/ml (2 pM). FGF repression occurs only during the G1 phase of the cell cycle by a mechanism that appears to be independent of ongoing cell proliferation. When exponentially growing myoblasts are deprived of FGF, cells become postmitotic within 2-3 h, express muscle-specific proteins within 6-7 h, and commence fusion within 12-14 h. Although expression of these three terminal differentiation phenotypes occurs at different times, all are initiated by a single regulatory "commitment" event in G1. The entire population commits to terminal differentiation within 12.5 h of FGF removal as all cells complete the cell cycle and move into G1. Differentiation does not require a new round of DNA synthesis. Comparison of MM14 behavior with other myoblast types suggests a general model for skeletal muscle development in which specific growth factors serve the dual role of stimulating myoblast proliferation and directly repressing terminal differentiation.

Myogenic defect in acetylcholinesterase regulation in muscular dystrophy of the chicken.

To determine whether inherited muscular dystrophy of the chicken is neurogenic or myogenic in origin, limb buds from homozygous normal and dystrophic chick embryos were exchanged prior to muscle differentiation and innervation. Biceps muscles of hatched chicks, in which muscle of the donor was innervated by nerves of the host, were analyzed for embryonic properties of muscle acetylcholinesterase and for fiber diameter, two distinctive markers for expression of the dystrophic gene. The results indicate that muscular dystrophy of the chicken is caused by an initial biochemical lesion in the limb and its muscle rather than in its innervating nerve.

Evidence that IGF-binding protein-5 functions as a growth factor.

Recent studies support the concept that IGF-binding protein-5 (IGFBP-5) stimulates bone formation, at least in part, via IGF-independent mechanisms. To evaluate this hypothesis further, we evaluated in vitro and in vivo effects of IGFBP-5 on bone formation parameters using the IGF-I knockout (KO) mouse. Treatment of serum-free cultures of osteoblast clones derived from IGF-I KO mice with recombinant human IGFBP-5 increased both proliferation and alkaline phosphatase (ALP) activity in a dose-dependent manner, an effect comparable to that seen with IGF-I. IGF-II levels from media conditioned by osteoblasts derived from IGF-I KO mouse were below those detectable by RIA. To eliminate possible actions of IGF-II, if any was produced by osteoblasts derived from IGF-I knockout mice, the IGFBP-5 effect was studied in the presence of exogenously added IGFBP-4, a potent inhibitor of IGF-II actions in bone cells. Addition of IGFBP-4 blocked IGF-I- but not IGFBP-5-induced cell proliferation in osteoblasts derived from IGF-I knockout mice. Consistent with in vitro results, a single local injection of IGFBP-5 to the outer periosteum of the parietal bone of IGF-I KO mice increased ALP activity and osteocalcin levels of calvarial bone extracts. The magnitudes of IGFBP-5-induced increases in ALP and osteocalcin in parietal bone extracts of IGF-I KO mice were comparable to those seen in C3H mice. In contrast to IGFBP-5, local administration of IGFBP-4 had no significant effect on bone formation in C3H and IGF-I KO mice. These results provide the first direct evidence to our knowledge that IGFBP-5 functions as a growth factor that stimulates its actions in part via an IGF-independent mechanism.

Copurification of osteolytic and transforming growth factor beta activities produced by human lung tumor cells associated with humoral hypercalcemia of malignancy.

The SK-Luci-6 cell line, established from a large-cell anaplastic lung tumor of a patient with humoral hypercalcemia of malignancy (HHM), was investigated to identify osteolytic factors produced that might mediate HHM. Most HHM-associated tumors are thought to produce parathyroid hormone-related proteins or transforming growth factor (TGF) alpha. SK-Luci-6 cells formed s.c. tumors and induced hypercalcemia in athymic nude mice. Serum-free conditioned medium from SK-Luci-6 cultures induced bone resorption in neonatal mouse calvariae in vitro, and also contained TGF-beta activity and mitogenic activity. SK-Luci-6 cell conditioned medium did not displace [125I]epidermal growth factor binding to cell receptors or stimulate cyclic AMP formation in rat osteosarcoma cells, suggesting that the conditioned medium did not contain TGF-alpha or parathyroid hormone-related proteins. The osteolytic, TGF-beta, and mitogenic activities copurified in several chromatographic separations: gel filtration in acid and then in guanidine HCl; ion exchange; and reverse phase. The results suggest that in the HHM-associated SK-Luci-6 tumor, the causative osteolytic factor produced by the tumor cells is not a parathyroid hormone-related protein or TGF-alpha but, rather, may be a TGF-beta.

Human prostatic cancer cells, PC3, elaborate mitogenic activity which selectively stimulates human bone cells.

Prostatic cancer typically produces osteoblastic metastases which are not attended by marrow fibrosis (i.e., osteoblast but not stromal fibroblast proliferation). In the present study we sought to test the hypothesis that prostatic cancer cells produce factor(s) which act selectively on human osteoblasts. Such a paracrine mechanism would explain the observed increase in osteoblasts, unaccompanied by an increase in marrow fibroblasts. To test this hypothesis we investigated the mitogenic activity released by the human prostatic tumor cell line, PC3. PC3 cells have been reported previously to produce mitogenic activity for cells that was relatively specific for rat osteoblasts compared to rat fibroblasts. However, the effects of this activity on human cells has not been examined previously. PC3-conditioned medium (CM) (5-50 micrograms CM protein/ml) stimulated human osteoblast proliferation by 200-950% yet did not stimulate human fibroblast proliferation [( 3H]thymidine incorporation). PC3 CM also increased cell numbers in human osteoblast but not fibroblast cell cultures. To determine whether the osteoblast-specific mitogenic activity could be attributed to known bone growth factors, specific assays for these growth factors were performed. PC3 CM contained 10 pg insulin-like growth factor (IGF) I, less than 2 pg IGF II, 54 pg basic fibroblast growth factor, and 16 pg transforming growth factor beta/microgram CM protein. None of these growth factors alone or in combination could account for the observed osteoblast-specific PC3 cell-derived mitogenic activity. Furthermore, when 5 micrograms/ml PC3 CM was tested in combination with maximally effective concentrations of either basic fibroblast growth factor, IGF I, IGF II, or transforming growth factor beta, it produced an additive effect suggesting that PC3 CM stimulates osteoblast proliferation by a mechanism independent of these bone mitogens. Biochemical characterization supported the hypothesis that the PC3 cell growth factor was unique from other growth factors. The PC3 growth factor did not bind to heparin and was resistant to acid as well as the reducing agent, dithiothreitol. Sephadex G-75 and fast protein liquid chromatography Mono S cation-exchange chromatography revealed the PC3-derived mitogen to be an Mr 26,000-30,000 basic protein. Therefore, we conclude that PC3 cells release a mitogen which exhibits higher specificity for human osteoblasts than human fibroblasts and is unique from other growth factors tested. Production of this mitogen by human prostatic carcinoma cells could play an etiological role in the intense osteoblast-specific stimulation that occurs at sites of bone metastases.

Primary structure of human skeletal growth factor: homology with human insulin-like growth factor-II.

Human skeletal growth factor (human SGF) extracted from human bone has been purified to homogeneity by hydroxyapatite chromatography and gel filtration under dissociative conditions followed by FPLC heparin-Sepharose affinity chromatography and reverse phase HPLC. Human SGF was homogeneous except that in each preparation about 30% of SGF molecules lacked the N-terminal alanine. 75% of the human SGF sequence has been determined. The amino acid sequences of the N-terminal 20 amino acids and of several tryptic fragments were identical to the corresponding sequences of human insulin-like growth factor-II (IGF-II) purified from serum. However, since the C-peptide (variable region) of human SGF has not yet been sequenced, we cannot conclude that SGF is identical to IGF-II. Comparison of the amino acid sequence of human SGF with that of IGF-II variants that have been described in the literature revealed that human SGF is not one of the known IGF-II variants. IGF-I was also found in human bone extract but was several-fold less abundant than SGF/IGF-II. The relative abundance of SGF/IGF-II and IGF-I in bone corresponded to the relative rates of production of these two mitogens by human bone cells in vitro. Regarding the physiological significance of IGF-II in bone, previous studies on the biological actions of SGF in vitro suggest that this growth factor can have both paracrine and autocrine functions on cells of the osteoblast line. In addition, we have proposed the concept that SGF is a mediator of the coupling of bone formation to bone resorption, an important bone volume regulatory mechanism. In as much as SGF is very similar (if not identical) to IGF-II, it seems likely that these proposed regulatory functions of SGF in bone are attributable to IGF-II.

Isolation and purification of a low-molecular-weight skeletal growth factor from human bones.

A low-molecular-weight potent bone cell mitogen termed human skeletal growth factor (human SGF) was purified to homogeneity from human bone matrix. Extraction and initial purification steps were done under dissociative conditions to separate human SGF from high-molecular-weight complexes of bone matrix proteins. SGF activity was extracted from human femoral heads by demineralization with 10% EDTA in the presence of 4 M guanidine-HCl and proteinase inhibitors and was purified by hydroxyapatite, HPLC gel-filtration and HPLC reverse-phase chromatography. Human SGF thus purified was homogeneous by HPLC reverse-phase chromatography and SDS-polyacrylamide gel electrophoresis. The relative molecular mass of human SGF purified under dissociative conditions was 11,000. Human SGF stimulated bone cell proliferation ([3H]thymidine incorporation and cell number) at picomolar concentrations, with half maximum activity at 2-3 ng/ml (180-270 pM). Human SGF constitutes 0.00024% of organic bone matrix by weight.

Structure and characterization of the human insulin-like growth factor binding protein (IGFBP)-6 promoter: identification of a functional retinoid response element.

The 1.7 kb human insulin-like growth factor binding protein (IGFBP)-6 gene 5'-flanking region was sequenced and found to have promoter activity in human osteoblasts. The sequence contains four clustered transcription start sites and three retinoic acid response elements (RAREs) with widely spaced half-sites. Only the proximal DR15 RARE was functional. Retinoids increased IGFBP-6 promoter activity up to 3-fold.

A mouse tumor-derived osteolytic factor stimulates bone resorption by a mechanism involving local prostaglandins production in bone.

Culture medium which was conditioned by tissue of a CE mouse breast tumor in vitro contained dose-dependent osteolytic activity. The osteolytic activity was not soluble in dichloromethane and ethylacetate, indicating that it was not attributable to vitamin D metabolites or prostaglandins. However, breast tumor-conditioned medium stimulated production and release of prostaglandin E2 from mouse calvaria in vitro, and the stimulation of bone resorption in vitro by breast tumor-conditioned medium was blocked by a dose of indomethacin that prevented stimulation of mouse calvarial prostaglandin E2 production and release. The resorptive activity of parathyroid hormone (PTH) was not affected by the same dose of indomethacin, suggesting that the osteolytic factor was not PTH. This was further supported by observation that mouse kidney cell cAMP production was stimulated by PTH, but not by the aqueous phase of ethylacetate-extracted breast tumor-conditioned medium. In addition to osteolytic activity, breast tumor-conditioned medium contained a dose-dependent bone cell mitogenic activity, demonstrated by the stimulation of [3H]thymidine incorporation into trichloroacetic acid-insoluble macromolecules and a corresponding increase in bone cell number in monolayer cultures of bone cells. Breast tumor-conditioned medium also contained a dose-dependent transforming growth factor-(TGF-) like activity as defined by its ability to transform anchorage-dependent growth of nontransformed cells to anchorage-independent growth. The TGF in breast tumor-conditioned medium did not compete with epidermal growth factor (EGF) for EGF receptor binding, but its transforming activity was greatly enhanced by EGF, indicating that it was a beta-type TGF. Both the osteolytic and mitogenic activities were nondialyzable, sensitive to reducing agent, and not removable by dichloromethane and ethylacetate extractions. Furthermore, the TGF activity was not removed by ethylacetate extraction. Thus, the possibility that these activities in breast tumor-conditioned medium might be mediated by the same molecule must be considered. In summary, our data suggest that the CE mouse mammary carcinoma cells produce and secrete into the culture medium an osteolytic factor which is neither PTH nor prostaglandin and which stimulates local synthesis in bone of prostaglandin E2 which in turn increases bone resorption in vitro.

Inhibitory insulin-like growth factor-binding protein: cloning, complete sequence, and physiological regulation.

In this study we report the preparation of a human osteosarcoma cell cDNA library and describe the isolation and sequence determination of a clone encoding the complete sequence of a novel human insulin-like growth factor (IGF)-binding protein (hIGFBP-4). Previous work indicated that hIGFBP-4 is the predominant IGFBP expressed by human osteoblast-like cells, and that IGFBP-4 binds and inhibits the mitogenic activities of IGF-I and IGF-II. Sequence determination revealed that hIGFBP-4 is a unique gene product with significant amino- and carboxy-terminal sequence similarity to three other known IGFBPs. Identical alignment of 18 cysteines in IGFBP-4 and the three other IGFBPs is a key structural feature of this protein family. In vitro studies of human osteoblast-like cells suggest that PTH regulates the expression of hIGFBP-4 and that the PTH effect is mediated through a cAMP mechanism. hIGFBP-4 mRNA was also expressed in skin fibroblasts, and thus, this inhibitory IGFBP could be an important physiological regulator of IGF actions in bone cells and other cell types as well.

Picomolar norethindrone in vitro stimulates the cell proliferation and activity of a human osteosarcoma cell line and increases bone collagen synthesis without an effect on bone resorption.

To determine how progestins increase bone formation in vivo, the effects of the synthetic progestin norethindrone (NET), on aspects of bone formation in vitro were determined. NET at picomolar concentrations in vitro stimulated the proliferation of human TE85 osteosarcoma cells as assessed by the increase in [3H]thymidine incorporation into DNA and in cell number and also stimulated the release of osteocalcin in both the presence and absence of 10 nM 1,25-(OH)2D3. NET increased cellular alkaline phosphatase specific activity (an index of osteoblastic differentiation), but at much higher concentrations, that is, nanomolar. These findings suggest that low concentrations of NET act directly on human TE85 osteosarcoma cells to stimulate their proliferation, differentiation, and cell activity. Furthermore, mitogenic doses of NET stimulated bone collagen synthesis both in a chicken calvarial organ culture assay (assessed by the incorporation and hydroxylation of [3H]proline) and in a human TE85 osteosarcoma cell culture assay (determined by the incorporation of [3H]proline into collagenase-digestible proteins). In contrast, NET at 10(-6)-10(-12) M had no apparent effect on the rate of basal or PTH-stimulated release of 45Ca from prelabeled mouse calvariae in vitro. In summary, this study has demonstrated for the first time that picomolar NET acted directly on human TE85 osteosarcoma cells to increase (1) cell proliferation and differentiation, (2) osteoblastic activity (i.e., osteocalcin synthesis), and (3) bone collagen synthesis in vitro. The same doses of NET in vitro did not reduce the bone resorption rate under our assay conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-6 messenger RNA expression and interleukin-6 protein secretion in cells isolated from normal human bone: regulation by interleukin-1.

Recent evidence suggests that cytokines, in addition to regulating hematopoiesis and immune functions, may be important paracrine regulators of bone turnover. Interleukin-1 (IL-1) and IL-6 are cytokines that are produced by and affect both hematopoietic and nonhematopoietic cell types. IL-1 stimulates bone resorption and inhibits osteoblast proliferation and collagen production. Previous reports that IL-6 was secreted in murine osteoblast and bone organ cultures in response to IL-1 and PTH suggested that IL-6 has paracrine effects on bone resorption or formation. To determine whether IL-6 has a paracrine function in human bone, IL-6 expression in cells isolated from normal human bone was investigated. IL-6 mRNA levels in untreated cultures were low and variable, and IL-6 secretion was undetectable. PTH had no effect on IL-6 mRNA levels or IL-6 secretion. IL-1 beta increased IL-6 mRNA levels, maximally 40-fold at 12 h. IL-1 beta increased IL-6 secretion to 0.13 nM, more than 80-fold that of untreated controls at 12 h. IL-1 beta also increased IL-1 beta mRNA levels, maximally 9-fold at 12 h, but did not increase cellular levels or secretion of IL-1 beta protein. Recombinant human IL-6 at 0.5-5 nM stimulated resorption in neonatal mouse calvarial organ cultures but had no effect on human bone-derived cell DNA synthesis or type I procollagen mRNA levels. The results suggest that IL-6 production by human osteoblasts may function to enhance osteolytic activity of IL-1 but does not affect proliferative and matrix biosynthetic aspects of bone formation that were tested. Because osteoblasts and bone marrow cells are in close proximity, IL-6 produced by osteoblasts may also function to amplify IL-1 stimulation of immune responses and hematopoiesis in bone marrow.

Autocrine regulators of MC3T3-E1 cell proliferation.

MC3T3-E1 cells, a clonal osteoblast-like mouse calvarial cell line, secrete several growth regulating factors. These regulators include insulin-like growth factor (IGF) type I, transforming growth factor beta (TGF-beta), and IGF-II in descending order of abundance. MC3T3-E1 cells in culture also produce two IGF binding proteins (IGFBP), M(r) 25 and 32 kD, having sequence identity with IGFBP-4 and IGFBP-6, respectively. In addition, this is the first observation that osteoblast-like bone cells in culture produce IGFBP-6. To determine if growth factors produced by MC3T3-E1 cells have autocrine actions on these cells, the effects of IGF-I, IGF II, TGF-beta 1, and IGFBP-4 on MC3T3-E1 cell proliferation were determined. Exogenous addition of IGF-I and IGF-II stimulated MC3T3-E1 cell proliferation, but TGF-beta 1 and IGFBP-4 inhibited MC3T3-E1 cell proliferation. Based on these findings, we conclude that MC3T3-E1 cells in culture produce autocrine regulators of MC3T3-E1 cell proliferation and that the actions of IGFs may also be regulated by IGFBPs produced by these same cells.

Insulinlike growth factor II and transforming growth factor beta regulate collagen expression in human osteoblastlike cells in vitro.

Insulinlike growth factor II (IGF-II) and transforming growth factor beta (TGF-beta) are the most abundant polypeptide growth factors found in human bone matrix and are produced by human bone cells in vitro. IGF-II and TGF-beta 1 increased total protein synthesis, collagenous protein synthesis, and the steady-state level of type I procollagen mRNA in a time-dependent manner in osteoblastlike cells isolated from human bone. Type III procollagen mRNA expression was low in untreated cultures and was not affected by IGF-II or TGF-beta. TGF-beta 1 elevated type I procollagen mRNA rapidly, with the maximal observed change at 10 h. In contrast, procollagen mRNA levels increased more slowly in response to IGF-II and reached a lower maximal level than with TGF-beta, but the response was sustained through 24 h. Collagenous protein synthesis in IGF-II- and TGF-beta-treated cells increased in parallel with increases in procollagen mRNA levels and was higher at 21 h for TGF-beta 1 and at 36 h for IGF-II. The difference in the time course and magnitude of change in type I procollagen mRNA levels in response to IGF-II and TGF-beta 1 suggests that these two growth factors work through distinct mechanisms that provide both a rapid transient response and a later sustained response in bone matrix biosynthetic activity.

Insulin-like growth factor (IGF)-I, -II, IGF binding proteins (IGFBP)-3, -4, and -5 levels in the conditioned media of normal human bone cells are skeletal site-dependent.

The skeleton in its function of affording strength and support to the body is subject to differential mechanical loading which has been implicated to mediate some of its effects on bone formation via the insulin-like growth factors (IGFs), which are important regulators of bone metabolism. We, therefore, sought to conduct the present study with the hypothesis that the skeletal site-dependent differences in mechanical loading and other variables including stage of osteoblast differentiation would be associated with site-specific differences in the production of the IGF system components. To test this hypothesis, conditioned media (CM) from normal human bone cells (control and IGF-II-treated 48-h cultures) from five different skeletal sites were obtained and assayed for IGF-I, IGF-II (following separation of IGF binding proteins [IGFBPs]), IGFBP-3, IGFBP-4, and IGFBP-5 protein levels employing specific radioimmunoassays for each protein. IGF-I levels were lower than any other IGF system component but were significantly different between the various sites tested. IGF-II levels were greatest in the CM from mandibular cells, followed by calvarial and rib cells, and least in the marrow stromal cells. IGFBP-3 levels were highest in the CM of vertebral cells and lowest in the CM of rib and mandibular cells. The relative abundance of IGFBP-4 in decreasing order was observed in mandibular, calvarial, vertebral, rib, and stromal cells' CM. IGFBP-5 was produced maximally by the calvarial cells, followed by the mandibular, vertebral, stromal, and rib cells. IGFBP-4 appeared to be the IGF system component most abundantly produced by all the cell types from the skeletal sites tested. On a molar basis, the IGFBPs in general were estimated to be produced at a higher magnitude than the IGFs. These findings indicate that there are skeletal site-dependent differences in the production of IGF system components and suggest that the regulation of bone metabolism may vary at the various skeletal sites.

Osteoclast formation in bone marrow cultures from two inbred strains of mice with different bone densities.

For the purpose of identifying genes that affect bone volume, we previously identified two inbred mouse strains (C57BL/6J and C3H/HeJ) with large differences in femoral bone density and medullary cavity volume. The lower density and larger medullary cavity volume in C57BL/6J mice could result from either decreased formation or increased resorption or both. We recently reported evidence suggesting that bone formation was increased in vivo and that osteoblast progenitor cells are more numerous in the bone marrow of C3H/HeJ compared with C57BL/6J mice. In the present study, we determined whether osteoclast numbers in vivo and osteoclast formation from bone marrow cells in vitro might also differ between the two mouse strains. We have found that the number of osteoclasts on bone surfaces of distal humerus secondary spongiosa was 2-fold higher in 5.5-week-old C57BL/6J mice than in C3H/HeJ mice of the same age (p < 0.001). Bone marrow cells of C57BL/6J mice cocultured with Swiss/Webster mouse osteoblasts consistently produced more osteoclasts than did C3H/HeJ bone marrow cells at all ages tested from 3.5-14 weeks of age (p < 0.001). Osteoclast formation was also greater from spleen cells of 3.5-week-old C57BL/6J mice than C3H/HeJ mice. The distribution of nuclei per osteoclast and the 1, 25-dihydroxyvitamin D3 dose dependence of osteoclast production from bone marrow cells were similar. Osteoclasts that developed from both C57BL/6J and C3H/HeJ marrow cells formed pits in dentin slices. Cultures from C57BL/6J marrow cells formed 2.5-fold more pits than cultures from C3H/HeJ marrow cells (p < 0.02). We compared the abilities of C57BL/6J and C3H/HeJ osteoblasts to support osteoclast formation. When bone marrow cells from either C57BL/6J or C3H/HeJ mice were cocultured with osteoblasts from either C57BL/6J or C3H/HeJ newborn calvaria, the strain from which osteoblasts were derived did not affect the number of osteoclasts formed from marrow cells of either strain. Together, these observations suggest that genes affecting the bone marrow osteoclast precursor population may contribute to the relative differences in bone density that occur between C3H/HeJ and C57BL/6J mouse strains.

Differential regulation of insulin-like growth factor-I (IGF-I) and IGF-II release from cultured neonatal mouse calvaria by parathyroid hormone, transforming growth factor-beta, and 1,25-dihydroxyvitamin D3.

In a previous study we found that PTH stimulated bone resorption and release of insulin-like growth factor-I (IGF-I) and IGF-II from cultured neonatal mouse calvaria. Since IGF-I and IGF-II stimulate osteoblast proliferation and collagen synthesis, these results suggested that increased release of IGFs during resorption could mediate in part coupling of bone formation to bone resorption. In the present study two other osteolytic agents, transforming growth factor-beta (TGF beta) and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3 were examined for effects on IGF release from neonatal mouse calvaria. Like PTH, TGF beta stimulated resorption and increased release of IGF-I and IGF-II. 1,25-(OH)2D3, however, stimulated resorption and IGF-II release comparable to PTH, but inhibited release of IGF-I. 1,25-(OH)2D3 (0.1-100 nM) inhibited basal release of IGF-I, and 10 nM 1,25-(OH)2D3 inhibited release of IGF-I induced by PTH or TGF beta. The effects of 1,25-(OH)2D3 were specific to this vitamin D metabolite and did not occur with 25-hydroxyvitamin D3 or 24,25-(OH)2D3 at the same concentration. Calcitonin (50 mU/ml) decreased 1,25-(OH)2D3 stimulation of resorption, but did not affect 1,25-(OH)2D3 stimulation of IGF-II release and inhibition of IGF-I release. This evidence that effects of 1,25-(OH)2D3 on release of the IGFs were independent of bone resorption supports the conclusion that 1,25-(OH)2D3 modulated the production and secretion of IGF-I and IGF-II in calvarial cells. The results of this and the previous study suggest that PTH, TGF beta, and 1,25-(OH)2D3 differentially regulate mouse calvarial cell IGF-I and IGF-II production.

Interleukin-1 stimulates release of insulin-like growth factor-I from neonatal mouse calvaria by a prostaglandin synthesis-dependent mechanism.

Interleukin-1 alpha (IL-1 alpha) and IL-1 beta are cytokines produced by cells of the immune system and nonimmune cells, such as osteoblasts. IL-1 alpha and IL-1 beta have potent stimulatory effects on bone resorption and also have mixed effects on bone formation, suggesting that they play an important role in local regulation of bone turnover. The present study examined paracrine mechanisms underlying effects of IL-1 on bone formation. Bone formation is regulated at the local level by polypeptide growth factors such as insulin-like growth factor-I (IGF-I). IGF-I is a relatively abundant growth factor in bone matrix, is produced by osteoblasts, and stimulates osteoblastic cell proliferation and bone matrix synthesis. Recent evidence suggests that IGF-I also stimulates osteoclast formation and bone resorption. To determine whether increased or decreased IGF-I production in bone might mediate some of the effects of IL-1 on bone turnover, the effects of IL-1 alpha and IL-1 beta on IGF-I release from neonatal mouse calvaria in organ culture were examined. IL-1 alpha and IL-1 beta stimulated resorption and increased the release of IGF-I into the medium during 6 days of culture. Maximal stimulation of resorption occurred at lower concentrations of IL-1 alpha and IL-1 beta (1 pM) than were required for maximum stimulation of IGF-I release (10 pM). IL-1 beta also increased steady state levels of 7.5- and 0.9-kilobase IGF-I mRNA transcripts in total RNA extracted from cultured calvaria at 24 h. The cyclooxygenase inhibitor indomethacin (1 microM) inhibited IL-1 beta-induced prostaglandin E2 (PGE2) production and inhibited resorption and release of IGF-I from calvaria. Indomethacin had a smaller effect on PTH-induced IGF-I release and had no significant effect on PTH-induced resorption. These results suggest that the effects of IL-1 on resorption and release of IGF-I may be mediated in part by a prostaglandin-dependent mechanism. Consistent with this hypothesis, PGE1 and PGE2 stimulated resorption and release of IGF-I. The dose responses for PGE2 stimulation of resorption and stimulation of IGF-I release were equivalent. The results are consistent with the hypotheses that 1) IL-1 stimulates IGF-I production by bone cells, in part by PG-dependent mechanisms; 2) the effects of IL-1 and PGs on bone formation and resorption may be mediated by locally induced secretion of IGF-I and other growth factors in bone.

Parathyroid hormone stimulates release of insulin-like growth factor-I (IGF-I) and IGF-II from neonatal mouse calvaria in organ culture.

Effects of increased bone resorption on release of insulin-like growth factor-I (IGF-I) and IGF-II into the osteoblast microenvironment were investigated using neonatal mouse calvaria organ cultures. Release of these growth factors from calvaria into serum-free medium was quantitated using a human IGF-I RIA and human IGF-II RRA. Untreated calvaria released several-fold more IGF-II than IGF-I. PTH (from 1-12 nM) stimulated a dose-dependent increase in the release of both growth factors that correlated with increased calcium release and was sustained for up to 6 days. IGF-I and IGF-II release were maximally stimulated 5- to 10-fold and 1.5- to 2-fold, respectively, compared to untreated control values. Calcitonin inhibited PTH-stimulated resorption, but had no effect on PTH stimulation of IGF-I and IGF-II release, suggesting that PTH effects on IGF-I and IGF-II release were not dependent on resorption. Furthermore, the amounts of IGF-I and IGF-II released from calvaria during 6 days of culture were 5-fold more than the amounts of IGF-I and IGF-II present in the calvaria (bone plus cells) at the beginning of culture, suggesting that much of the IGF-I and IGF-II released was newly produced by calvaria cells. The results suggest that PTH directly stimulated calvarial osteoblasts to release IGF-I and IGF-II. Since IGF-I and IGF-II stimulate osteoblastic cell proliferation, the effect of PTH on the release of these and other growth factors may mediate coupling of bone formation to bone resorption.

Fibroblast growth factor enhances human chorionic gonadotropin synthesis independent of mitogenic stimulation in Jar choriocarcinoma cells.

The Jar choriocarcinoma cell line was used as an in vitro placental cell model to determine the effects of polypeptide growth factors on hCG beta secretion. Epidermal and fibroblast growth factor (FGF) treatment of serum-free cultures stimulated hCG beta secretion 2.5- and 4.0-fold over basal serum-free control levels within a 15-h incubation period. Insulin-like growth factor I, nerve growth factor, and transforming growth factor-beta had no significant effect on hCG beta secretion. FGF at concentrations as low as 0.125 ng/ml significantly elevated medium hCG beta levels without increasing cell number or total cellular protein. FGF stimulation of secretion was not detectable until 2 h of treatment. Intracellular hCG beta remained constant (23%) relative to total hCG beta (cell plus medium) as total hCG beta increased 3-fold, suggesting that FGF stimulated de novo hCG beta synthesis. Insulin significantly augmented the FGF-induced hCG beta stimulation without stimulating hCG beta production itself.