Increased osteoclast development after estrogen loss: mediation by interleukin-6.

Osteoclasts, the cells that resorb bone, develop from hematopoietic precursors of the bone marrow under the control of factors produced in their microenvironment. The cytokine interleukin-6 can promote hematopoiesis and osteoclastogenesis. Interleukin-6 production by bone and marrow stromal cells is suppressed by 17 beta-estradiol in vitro. In mice, estrogen loss (ovariectomy) increased the number of colony-forming units for granulocytes and macrophages, enhanced osteoclast development in ex vivo cultures of marrow, and increased the number of osteoclasts in trabecular bone. These changes were prevented by 17 beta-estradiol or an antibody to interleukin-6. Thus, estrogen loss results in an interleukin-6-mediated stimulation of osteoclastogenesis, which suggests a mechanism for the increased bone resorption in postmenopausal osteoporosis.

Interleukin-11: a new cytokine critical for osteoclast development.

Stromal cells of the bone marrow control the development of osteoclasts through the production of cytokines capable of promoting the proliferation and differentiation of hematopoietic progenitors. Moreover, the deregulated production of the cytokine IL-6 in the bone marrow mediates an increase in osteoclastogenesis after estrogen loss. IL-6, however, does not influence osteoclastogenesis in the estrogen-replete state, suggesting that other cytokines might be responsible for osteoclast development under physiologic circumstances. We report here that IL-11, a newly discovered cytokine that is produced by marrow stromal cells, induced the formation of osteoclasts exhibiting an unusually high degree of ploidy in cocultures of murine bone marrow and calvarial cells. Osteoclasts formed in the presence of IL-11 were capable of bone resorption, as evidenced by the formation of resorption pits, as well as the release of 45Ca from prelabeled murine calvaria. Further, an antibody neutralizing IL-11 suppressed osteoclast development induced by either 1,25-dihydroxyvitamin D3, parathyroid hormone, interleukin-1, or tumor necrosis factor; whereas inhibitors of IL-1 or TNF had no effect on IL-11-stimulated osteoclast formation. The effects of IL-11 on osteoclast development were blocked by indomethacin; more important, however, they were independent of the estrogen status of the marrow donors.

Inhibition of osteoblastogenesis and promotion of apoptosis of osteoblasts and osteocytes by glucocorticoids. Potential mechanisms of their deleterious effects on bone.

Glucocorticoid-induced bone disease is characterized by decreased bone formation and in situ death of isolated segments of bone (osteonecrosis) suggesting that glucocorticoid excess, the third most common cause of osteoporosis, may affect the birth or death rate of bone cells, thus reducing their numbers. To test this hypothesis, we administered prednisolone to 7-mo-old mice for 27 d and found decreased bone density, serum osteocalcin, and cancellous bone area along with trabecular narrowing. These changes were accompanied by diminished bone formation and turnover, as determined by histomorphometric analysis of tetracycline-labeled vertebrae, and impaired osteoblastogenesis and osteoclastogenesis, as determined by ex vivo bone marrow cell cultures. In addition, the mice exhibited a threefold increase in osteoblast apoptosis in vertebrae and showed apoptosis in 28% of the osteocytes in metaphyseal cortical bone. As in mice, an increase in osteoblast and osteocyte apoptosis was documented in patients with glucocorticoid-induced osteoporosis. Decreased production of osteoclasts explains the reduction in bone turnover, whereas decreased production and apoptosis of osteoblasts would account for the decline in bone formation and trabecular width. Furthermore, accumulation of apoptotic osteocytes may contribute to osteonecrosis. These findings provide evidence that glucocorticoid-induced bone disease arises from changes in the numbers of bone cells.

Increased bone formation by prevention of osteoblast apoptosis with parathyroid hormone.

The mass of regenerating tissues, such as bone, is critically dependent on the number of executive cells, which in turn is determined by the rate of replication of progenitors and the life-span of mature cells, reflecting the timing of death by apoptosis. Bone mass can be increased by intermittent parathyroid hormone (PTH) administration, but the mechanism of this phenomenon has remained unknown. We report that daily PTH injections in mice with either normal bone mass or osteopenia due to defective osteoblastogenesis increased bone formation without affecting the generation of new osteoblasts. Instead, PTH increased the life-span of mature osteoblasts by preventing their apoptosis - the fate of the majority of these cells under normal conditions. The antiapoptotic effect of PTH was sufficient to account for the increase in bone mass, and was confirmed in vitro using rodent and human osteoblasts and osteocytes. This evidence provides proof of the basic principle that the work performed by a cell population can be increased by suppression of apoptosis. Moreover, it suggests novel pharmacotherapeutic strategies for osteoporosis and, perhaps, other pathologic conditions in which tissue mass diminution has compromised functional integrity.

Attenuation of the self-renewal of transit-amplifying osteoblast progenitors in the murine bone marrow by 17 beta-estradiol.

In agreement with evidence that estrogens slow the rate of bone remodeling by suppressing the production of both osteoclasts and osteoblasts, loss of estrogens leads to an increase in the number of osteoclast as well as early osteoblast progenitors (CFU-osteoblasts; CFU-OBs) in the murine bone marrow. Here we show that CFU-OBs are early transit-amplifying progenitors, i.e., dividing cells capable of limited self-renewal, and that 17 beta-estradiol acts in vivo and in vitro to attenuate their self-renewal by approximately 50%. Consistent with a direct receptor-mediated action of estrogens on early mesenchymal cell progenitors, anti-estrogen receptor-alpha (anti-ER alpha) Ab's stain a small number of marrow cells that exhibit characteristics of primitive undifferentiated cells, including a high nucleus/cytoplasm ratio and lack of lineage-specific biochemical markers; the effect of 17 beta-estradiol on CFU-OB self-renewal is absent in mice lacking ER alpha. Because both osteoblasts and the stromal/osteoblastic cells that are required for osteoclast development are derived from CFU-OBs, suppression of the self-renewal of this common progenitor may represent a key mechanism of the anti-remodeling effects of estrogens.

Loss of estrogen upregulates osteoblastogenesis in the murine bone marrow. Evidence for autonomy from factors released during bone resorption.

Loss of sex steroids causes an increase in both the resorption and formation of bone, with the former exceeding the latter. Based on evidence that the increased bone resorption after estrogen loss is due to an increase in osteoclastogenesis, we hypothesized that estrogen loss also stimulates osteoblastogenesis. We report that the number of mesenchymal osteoblast progenitors in the murine bone marrow was increased two- to threefold between 2 and 8 wk after ovariectomy and returned to control levels by 16 wk. Circulating osteocalcin, as well as osteoclastogenesis and the rate of bone loss, followed a very similar temporal pattern. Inhibition of bone resorption by administration of the bisphosphonate alendronate led to a decrease of the absolute number of osteoblast progenitors; however, it did not influence the stimulating effect of ovariectomy on osteoblastogenesis or osteoclastogenesis. These observations indicate that the increased bone formation that follows loss of estrogen can be explained, at least in part, by an increase in osteoblastogenesis. Moreover, they strongly suggest that unlike normal bone remodeling, whereby osteoblast development is stimulated by factors released from the bone matrix during osteoclastic resorption, estrogen deficiency unleashes signals that can stimulate the differentiation of osteoblast progenitors in a fashion that is autonomous from the need created by bone resorption, and therefore, inappropriate.

Linkage of decreased bone mass with impaired osteoblastogenesis in a murine model of accelerated senescence.

Bone marrow is the principal site for osteoclastogenesis and osteoblastogenesis; and an increase in the former has been linked with bone loss caused by acute loss of gonadal steroids. We have now used an established murine model of accelerated senescence and osteopenia (SAMP6) to test the hypothesis that reduced osteoblastogenesis is linked with decreased bone mass. At 1 mo of age, the number of osteoblast progenitors in SAMP6 marrow was indistinguishable from controls; however a threefold decrease was found at 3-4 mo of age. Impaired osteoblast formation was temporally associated with decreased bone formation and decreased bone mineral density, as determined by histomorphometric analysis of tetracycline-labeled cancellous bone and dual-energy x-ray absorptiometry, respectively. Osteoclastogenesis determined in ex vivo bone marrow cultures was also decreased in these mice, as was the number of osteoclasts in histologic sections. Moreover, unlike controls, senescence-accelerated mice failed to increase osteoclast development after gonadectomy. The osteoclastogenesis defeat was secondary to impaired osteoblast formation as evidenced by the fact that osteoclastogenesis could be restored by addition of osteoblastic cells from normal mice. These findings provide the first demonstration of a link between low bone mineral density and decreased osteoblastogenesis in the bone marrow and validate the senescence-accelerated mouse as a model of involutional osteopenia.

Parathyroid hormone and lipopolysaccharide induce murine osteoblast-like cells to secrete a cytokine indistinguishable from granulocyte-macrophage colony-stimulating factor.

Osteoblasts are the cells responsible for the secretion of collagen and ultimately the formation of new bone. These cells have also been shown to regulate osteoclast activity by the secretion of cytokines, which remain to be defined. In an attempt to identify these unknown cytokines, we have induced primary murine osteoblasts with two bone active agents, parathyroid hormone (PTH) and lipopolysaccharide (LPS) and analyzed the conditioned media (CM) for the presence of specific cytokines. Analysis of the CM was accomplished by functional, biochemical, and serological techniques. The data indicate that both PTH and LPS are capable of inducing the osteoblasts to secrete a cytokine, which by all of the techniques used, is indistinguishable from granulocyte-macrophage colony-stimulating factor (GM-CSF). Secretion of GM-CSF is not constitutive and requires active induction. Production of the cytokine is dependent on the dose of PTH or LPS added. It has been demonstrated that the addition of GM-CSF to bone marrow cultures results in the formation of increased numbers of osteoclasts. Therefore, these data suggest that osteoblasts not only participate in bone remodeling by formation of new matrix but may regulate osteoclast activity indirectly by their ability to regulate hematopoiesis.

17 beta-estradiol inhibits interleukin-6 production by bone marrow-derived stromal cells and osteoblasts in vitro: a potential mechanism for the antiosteoporotic effect of estrogens.

The effect of 17 beta-estradiol on interleukin-6 (IL-6) synthesis was examined in murine bone marrow-derived stromal cell lines, normal human bone-derived cells, and nontransformed osteoblast cell lines from mice and rats. In all these cell types IL-6 production was stimulated as much as 10,000-fold in response to the combination of recombinant interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha). Addition of 17 beta-estradiol in the cultures exerted a dose-dependent inhibition of IL-1-, TNF-, and IL-1 + TNF-induced production of bioassayable IL-6. Testosterone and progesterone (but not 17 alpha-estradiol) also inhibited IL-6, but their effective concentrations were two orders of magnitude higher than 17 beta-estradiol. 17 beta-estradiol also decreased the levels of the IL-6 mRNA. In addition, estradiol inhibited both TNF-induced IL-6 production and osteoclast development in primary bone cell cultures derived from neonatal murine calvaria. The TNF-stimulated osteoclast development was also suppressed by a neutralizing monoclonal anti-IL-6 antibody. This in vitro evidence suggests, for the first time, a mechanistic paradigm by which estrogens might exert at least part of their antiresorptive influence on the skeleton.

Regulation of interleukin-6, osteoclastogenesis, and bone mass by androgens. The role of the androgen receptor.

Interleukin-6 is an essential mediator of the bone loss caused by loss of estrogens. Because loss of androgens also causes bone loss, we have examined whether the IL-6 gene is regulated by androgens, and whether IL-6 plays a role in the bone loss caused by androgen deficiency. Both testosterone and dihydrotestosterone inhibited IL-6 production by murine bone marrow-derived stromal cells. In addition, testosterone, dihydrotestosterone, and adrenal androgens inhibited the expression of a chloramphenicol acetyl transferase reporter plasmid driven by the human IL-6 promoter in HeLa cells cotransfected with an androgen receptor expression plasmid; however, these steroids were ineffective when the cells were cotransfected with an estrogen receptor expression plasmid. In accordance with the in vitro findings, orchidectomy in mice caused an increase in the replication of osteoclast progenitors in the bone marrow which could be prevented by androgen replacement or administration of an IL-6 neutralizing antibody. Moreover, bone histomorphometric analysis of trabecular bone revealed that, in contrast to IL-6 sufficient mice which exhibited increased osteoclast numbers and bone loss following orchidectomy, IL-6 deficient mice (generated by targeted gene disruption) did not. This evidence demonstrates that male sex steroids, acting through the androgen-specific receptor, inhibit the expression of the IL-6 gene; and that IL-6 mediates the upregulation of osteoclastogenesis and therefore the bone loss caused by androgen deficiency, as it does in estrogen deficiency.

The effect of calcium ion transport ATPase upon the passive calcium ion permeability of phospholipid vesicles.

The uptake and release of Ca2+ by sarcoplasmic reticulum fragments and reconstituted ATPase vesicles was measured by a stopped-flow fluorescence method using chlortetracycline as Ca2+ indicator. Incorporation of the Ca2+ transport ATPase into phospholipid bilayers of widely different fatty acid composition increases their passive permeability to Ca2+ by several orders of magnitude. Therefore in addition to participating in active Ca2+ transport, the (Mg2+ + Ca2+)-activated ATPase also forms hydrophilic channels across the membrane. The relative insensitivity of the permeability effect of ATPase to changes in the fatty acid composition of the membrane is in accord with the suggestion that the Ca2+ channels arise by protein-protein interaction between four ATPase molecules. The reversible formation of these channels may have physiological significance in the rapid Ca2+ release from the sarcoplasmic reticulum during activation of muscle.

Chronic elevation of parathyroid hormone in mice reduces expression of sclerostin by osteocytes: a novel mechanism for hormonal control of osteoblastogenesis.

Both chronic excess of PTH, as in hyperparathyroidism, and intermittent elevation of PTH (by daily injections) increase the number of osteoblasts; albeit, the former is associated with bone catabolism and the later with bone anabolism. Intermittent PTH increases osteoblast number by attenuating osteoblast apoptosis, an effect that requires the transcription factor Runx2. However, chronic elevation of PTH does not affect osteoblast apoptosis because it stimulates the proteasomal degradation of Runx2. Here, we studied the effects of PTH on Sost, a Runx2 target gene expressed in osteocytes (former osteoblasts embedded in the bone matrix), which antagonizes the pro-osteoblastogenic actions of bone morphogenetic proteins and Wnts. We report that continuous infusion of PTH to mice for 4 d decreased Sost mRNA expression in vertebral bone by 80-90%. This effect was accompanied by a comparable reduction of sclerostin, the product of Sost, in osteocytes, as determined by quantitative immunoblot analysis of bone extracts and by immunostaining. In contrast, a single injection of PTH caused a transient 50% reduction in Sost mRNA at 2 h, but four daily injections had no effect on Sost mRNA or sclerostin. PTH strongly decreased Sost expression in osteocytes formed in primary cultures of neonatal murine calvaria cells as well as in osteocytic MLO-A5 cells, demonstrating a direct effect of PTH on this cell type. These results, together with evidence that sclerostin antagonizes bone morphogenetic proteins and Wnts, strongly suggest that suppression of Sost by PTH represents a novel mechanism for hormonal control of osteoblastogenesis mediated by osteocytes.

Estrogen loss upregulates hematopoiesis in the mouse: a mediating role of IL-6.

We have previously demonstrated that ovariectomy causes an increase in the number of colony-forming unit granulocyte/macrophage (CFU-GM) and an upregulation of osteoclastogenesis in mice, both of which are mediated by interleukin-6 (IL-6). IL-6 is involved in the development of several hematopoietic progenitors, including the burst-forming unit-erythroid (BFU-E) and multipotent CFUs (CFU-GEMM). Therefore, we performed studies to examine if other hematopoietic progenitors, besides CFU-GM and their progeny, are affected by estrogen loss. We found that ovariectomy caused an increase in the number of CFU-GEMM and BFU-E, as well as an increase of CFU-GM in marrow cells of the femur. Administration of 17 beta-estradiol or a neutralizing antibody against IL-6 prevented the ovariectomy-induced increase in the number of these progenitors in the marrow. Ovariectomy also caused an increase in the number of circulating lymphocytes, neutrophils, and monocytes, which were suppressed by administration of 17 beta-estradiol or the neutralizing antibody against IL-6; however, the number of circulating platelets was unaffected by loss of ovarian function. These data establish that, in addition to upregulation of osteoclastogenesis, loss of estrogens in the mouse causes widespread effects on hematopoiesis, which are apparently mediated by IL-6.

Osteoblast programmed cell death (apoptosis): modulation by growth factors and cytokines.

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.

Chromosomal mapping of osteopenia-associated quantitative trait loci using closely related mouse strains.

Peak bone mineral density (BMD) is a highly heritable trait in humans and is currently the best predictor of skeletal fragility underlying osteoporosis. The SAMP6 mouse strain displays unusually low BMD at maturity, and age-dependent osteopenia associated with defective osteoblastogenesis. To identify quantitative trait loci (QTLs) influencing bone density, we constructed crosses between SAMP6 and either AKR/J or SAMP6, two related mouse strains of higher peak BMD. Due to common ancestry of these strains, intercross parents differed at only 39-40% of 227 highly-polymorphic genotyping markers, thus restricting our search to this informative portion of the genome and reducing the number of mice required for QTL significance. Using dual energy X-ray absorptiometry (DEXA), we measured spinal BMD in F2 cross progeny at 4 months of age, and selectively genotyped those in the highest and lowest quartiles for BMD. Based on linear regression of bone density on genotype, including Composite Interval Mapping to enhance mapping precision while adjusting for effects of distal markers, we identified multiple QTLs significantly affecting spinal BMD; these were mapped to regions of chromosomes 2 (two sites, one confirmed in both crosses), 7, 11, 13 and 16. One of these loci had been previously identified as a significant bone-density QTL, while 3 substantiate QTLs suggested by a low-power study of 24 recombinant-inbred mouse lines. Such recurrent appearance of QTLs, especially in crosses involving distantly-related strains, implies that polymorphism at these loci may be favored by evolution and might underlie variation in peak bone density among humans.

Essential requirement of BMPs-2/4 for both osteoblast and osteoclast formation in murine bone marrow cultures from adult mice: antagonism by noggin.

Bone morphogenetic proteins (BMPs) have been heretofore implicated in the induction of osteoblast differentiation from uncommitted progenitors during embryonic skeletogenesis and fracture healing. We have tested the hypothesis that BMPs are also involved in the osteoblastogenesis that takes place in the bone marrow in postnatal life. To do this, we took advantage of the properties of noggin, a recently discovered protein that binds BMP-2 and -4 and blocks their action. Addition of human recombinant noggin to bone marrow cell cultures from normal adult mice inhibited both osteoblast and osteoclast formation; these effects were reversed by exogenous BMP-2. Consistent with these findings, BMP-2 and -4 and BMP-2/4 receptor transcripts and proteins were detected in these primary cultures, in a bone marrow-derived stromal/osteoblastic cell line, as well as in murine adult whole bone; noggin expression was also documented in all these preparations. Moreover, addition of antinoggin antibody caused an increase in osteoblast progenitor formation. These findings suggest that BMP-2 and -4 are expressed in the bone marrow in postnatal life and serve to maintain the continuous supply of osteoblasts and osteoclasts; and that, in fact, BMP-2/4-induced commitment to the osteoblastic lineage is a prerequisite for osteoclast development. Hence, BMPs, perhaps in balance with noggin and possibly other antagonists, may provide the tonic baseline control of the rate of bone remodeling on which other inputs (e.g., hormonal, biomechanical, etc.) operate.

Increased adipogenesis and myelopoiesis in the bone marrow of SAMP6, a murine model of defective osteoblastogenesis and low turnover osteopenia.

Bone formation and hematopoiesis are anatomically juxtaposed and share common regulatory mechanisms. However, little is known about the interrelationship between these two processes. We have previously shown that the senescence accelerated mouse-P6 (SAMP6) exhibits decreased osteoblastogenesis in the bone marrow that is temporally linked with a low rate of bone formation and decreased bone mineral density. Here we report that in contrast to decreased osteoblastogenesis, ex vivo bone marrow cultures from SAMP6 mice exhibited an increase in the number of colony-forming unit adipocytes, as well as an increase in the number of fully differentiated marrow adipocytes, compared with SAMR1 (nonosteopenic) controls. Further, long-term bone marrow cultures from SAMP6 produced an adherent stromal layer more rapidly, generated significantly more myeloid progenitors and produced more IL-6 and colony-stimulating activity. Consistent with this, the number of myeloid cells in freshly isolated marrow from SAMP6 mice was increased, as was the number of granulocytes in peripheral blood. The evidence that SAMP6 mice exhibit decreased osteoblastogenesis, and increased adipogenesis and myelopoiesis, strongly suggests that a switch in the differentiation program of multipotential mesenchymal progenitors may underlie the abnormal phenotype manifested in the skeleton and other tissues of these animals. Moreover, these observations support the contention for the existence of a reciprocal relationship between osteoblastogenesis and adipogenesis that may explain the association of decreased bone formation and the resulting osteopenia with the increased adiposity of the marrow seen with advancing age in animals and humans.

Quantification of vitamin D receptor mRNA by competitive polymerase chain reaction in PBMC: lack of correspondence with common allelic variants.

It has been recently claimed that polymorphism for the vitamin D receptor (VDR) influences several aspects of calcium and bone metabolism. To evaluate the physiologic plausibility of these claims, we compared the abundance of the VDR mRNA in peripheral blood mononuclear cells (PBMCs) between different VDR genotypes using a quantitative reverse transcribed polymerase chain reaction-based method. The method is based on the coamplification of VDR cDNA and an internal standard consisting of known concentrations of a human VDR CDNA mutated at a BglII restriction site; the interassay coefficient of variation is 11%. To validate the method, we made use of earlier receptor binding studies indicating that normal human monocytes and activated, but not resting, lymphocytes expressed the VDR. The concentration of the VDR mRNA was 10(-8) to 10(-7) g/g of total RNA in cell-sorted monocytes and in in vitro activated lymphocytes, but only 10(-12) g/g of total mRNA in resting lymphocytes, establishing that the VDR mRNA determined by our method in PBMCs is due to constitutive expression in monocytes. Following an initial genotype screening of 85 normal volunteers by polymerase chain reaction or restriction fragment length polymorphism analysis, 14 individuals with the Bb genotype, 12 with the bb genotype, and 12 with the BB genotype were selected. The concentration of the VDR mRNA, corrected for the number of monocytes, was similar among the three genotype groups, as were the other variables examined: serum calcitriol, serum osteocalcin, and vertebral and hip bone density. We conclude that VDR polymorphism does not affect the abundance of the VDR mRNA.

Role of phosphodiesterase in the parathormone-stimulated adenosine 3',5'-monophosphate response in bone cell populations enriched in osteoclasts and osteoblasts.

The stimulation of cAMP formation by parathormone was assessed in isolated bone cell populations enriched in either osteoclast-like (CL) or osteoblast-like (BL) cells. In the presence of the potent phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), at 37 C, the CL cells produced at least 20% the amount of cAMP formed by the BL cells, but only about 5% when the inhibitor was omitted. The rate of cAMP production in response to parathormone was measured at 23 C in order to obtain a better indication of the capacity of the cells to generate this nucleotide. Under these conditions, the CL cells produced cAMP at a rate of 50-60% that of the BL cells regardless of whether IBMX was present. The phosphodiesterase activity in CL cell extracts was twice that in BL cell extracts, and IBMX inhibited this activity. These results show that when an effective inhibitor of phosphodiesterase is present, the CL cells exhibit a significant response to parathormone in terms of cAMP production. The possibility that the cAMP response in the CL cells was due to contaminating BL cells (which would have to be about 50% of the total cell number) was ruled out by the demonstration that citrate decarboxylating activity, a marker for the BL cells, was low and was not inhibited by parathormone. These data reinforce the earlier contention that parathormone directly stimulates adenylate cyclase in both osteoclasts and osteoblasts and activates bone resorption via cAMP as second messenger.

A collagenolytic response to parathormone, 1,25-dihydroxycholecalciferol D3, and prostaglandin E2 in bone of osteopetrotic (mi/mi) mice.

Stimulators of bone resorption, such as PTH, 1,25-dihydroxycholecalciferol [1,25-(OH)2D3], or prostaglandin E2 (PGE2), do not cause calcium release from cultured calvaria of the genetically determined osteopetrotic microphthalmic (mi/ mi) mouse, due to a defect in the function of osteoclasts. To investigate the capacity of cells of mi/mi bone to degrade collagen, calvaria of 1- to 3-day-old normal and mi/mi littermates were labeled in vivo with [3H]proline 16 h before removal, followed by culture in resorption medium. PTH, 1,25-(OH)2D3, and PGE2 stimulated the release of 3H-labeled material into the culture medium from both normal and mi/mi calvaria. The labeled substance released was of collagenous origin, as indicated by its content of hydroxyproline and susceptibility to collagenase. PTH also stimulated the release of 3H-labeled materials from normal calvaria labeled in vivo 112 h before the mice were killed, but had little or no effect on 3H release from the mi/mi bone, indicating that only noncalcified collagen is susceptible to hormone-stimulated degradation in osteopetrotic bone. We conclude that a portion of the hormone-stimulated resorptive mechanism, namely collagenolysis, is functional in bone of mi/mi mice. This result helps to pinpoint the resorptive defect in mi/ mi bone to a failure to dissolve mineral, rather than a more general phenomenon of failure to remove both mineral and matrix.