Screening of subfertile men for testicular carcinoma in situ by an automated image analysis-based cytological test of the ejaculate.

Testicular cancer (TC) is usually diagnosed after manifestation of an overt tumour. Tumour formation is preceded by a pre-invasive and asymptomatic stage, carcinoma in situ (CIS) testis, except for very rare subtypes. The CIS cells are located within seminiferous tubules but can be exfoliated and detected in ejaculates with specific CIS markers. We have built a high throughput framework involving automated immunocytochemical staining, scanning microscopy and in silico image analysis allowing automated detection and grading of CIS-like stained objects in semen samples. In this study, 1175 ejaculates from 765 subfertile men were tested using this framework. In 5/765 (0.65%) cases, CIS-like cells were identified in the ejaculate. Three of these had bilateral testicular biopsies performed and CIS was histologically confirmed in two. In total, 63 bilateral testicular biopsy were performed in conjunction with analysis of the ejaculates because of infertility work-up. Histological analysis of the biopsies for the presence of CIS yielded a test sensitivity of 0.67 and a specificity of 0.98. In addition, ejaculates from 45 patients with clinical signs of an overt TC were investigated and yielded a slightly lower sensitivity (0.51), possibly because of obstruction. We conclude that this novel non-invasive test combining automated immunocytochemistry and advanced image analysis allows identification of TC at the CIS stage with a high specificity, but a negative test does not completely exclude CIS. On the basis of the results, we propose that the assay could be offered to subfertile men and other patients who are at increased risk of TC.

The type I collagen fragments ICTP and CTX reveal distinct enzymatic pathways of bone collagen degradation.

Bone resorption may generate collagen fragments such as ICTP and CTX, which can be quantified in serum and/or urine by using specific immunoassays, and which are used as clinical markers. However, the relative abundance of ICTP and CTX varies according to the type of bone pathology, suggesting that these two fragments are generated through distinct collagenolytic pathways. In this study, we analyzed the release of ICTP and CTX from bone collagen by the proteinases reported to play a role in the solubilization of bone matrix. Cathepsin K released large amounts of CTX, but did not allow a detectable release of ICTP. Conversely, the matrix metalloproteinases (MMPs) MMP-2, -9, -13, or -14 released ICTP, but did not allow a detectable release of CTX. Next we analyzed the release of ICTP and CTX from bone explants cultured in the presence of well-established inhibitors of these proteinases and of matrix solubilization. An inhibitor of cysteine proteinases including cathepsin K, inhibited the release of CTX, but not the release of ICTP. MMP inhibitors inhibited the release of ICTP, but also that of CTX, in agreement with the putative role of MMPs in the initiation of bone resorption in addition to matrix solubilization. Similarly the treatment of mice bearing bone metastasis with an MMP inhibitor led to a significant reduction of serum ICTP and CTX, and osteolytic lesions. We conclude that the generation of ICTP and CTX depends on different collagenolytic pathways. This finding may explain why these two markers may discriminate between different bone pathologies.

Transforming growth factor-beta-induced osteoblast elongation regulates osteoclastic bone resorption through a p38 mitogen-activated protein kinase- and matrix metalloproteinase-dependent pathway.

Transforming growth factor-beta (TGF-beta) is a powerful modulator of bone metabolism, and both its anabolic and catabolic effects on bone have been described. Here we have tested the hypothesis that TGF-beta-induced changes in osteoblast shape promote bone resorption by increasing the surface area of bone that is accessible to osteoclasts. The addition of TGF-beta1 to MC3T3-E1 cells resulted in cytoskeletal reorganization, augmented expression of focal adhesion kinase, and cell elongation, accompanied by an increase in the area of cell-free substratum. TGF-beta1 also triggered activation of Erk1/2 and p38 mitogen-activated protein (MAP) kinase. The p38 MAP kinase inhibitor PD169316, but not an inhibitor of the Erk1/2 pathway, abrogated the effect of TGF-beta1 on cell shape. The matrix metalloproteinase inhibitor GM6001 also interfered with osteoblast elongation. Treatment of MC3T3-E1 cells seeded at confluence onto bone slices to mimic a bone lining cell layer with TGF-beta1 also induced cell elongation and increased pit formation by subsequently added osteoclasts. These effects were again blocked by PD169316 and GM6001. We propose that this novel pathway regulating osteoblast morphology plays an important role in the catabolic effects of TGF-beta on bone metabolism.

Phosphinic peptide inhibitors of macrophage metalloelastase (MMP-12). Selectivity and mechanism of binding.

Pseudopeptide inhibitors of MMP-12 with a phosphinic dipeptide G psi[PO(2)H-CH(2)]L covering the P1-P1'- positions originating from a combinatorial solid phase library have been identified and kinetically analysed with respect to binding mechanism and selectivity towards MMP-7, MMP-9, MMP-13 and MMP-14. One compound with a low nanomolar dissociation constant for MMP-12 showed significantly lower affinity towards all other MMPs tested compared to MMP-12. Two compounds showed selectivity against MMP-9, MMP-13 and MMP-14. One additional compound showed selectivity against MMP-7. The selectivity of these compounds could partly be rationalized by analysis of homology models of the enzymes. Truncated versions of one inhibitor spanning P2 to P2', P3 to P2' or P2 to P3' showed that interactions on both the prime and the non-prime side are important for binding. A two-step binding mechanism, with a rate limiting second step, was shown for binding of a tryptophane containing inhibitor to MMP-12 by transient state analysis, using the tryptophane residue of the inhibitor as fluorescent probe.

Matrix metalloproteinase 9 and vascular endothelial growth factor are essential for osteoclast recruitment into developing long bones.

Bone development requires the recruitment of osteoclast precursors from surrounding mesenchyme, thereby allowing the key events of bone growth such as marrow cavity formation, capillary invasion, and matrix remodeling. We demonstrate that mice deficient in gelatinase B/matrix metalloproteinase (MMP)-9 exhibit a delay in osteoclast recruitment. Histological analysis and specialized invasion and bone resorption models show that MMP-9 is specifically required for the invasion of osteoclasts and endothelial cells into the discontinuously mineralized hypertrophic cartilage that fills the core of the diaphysis. However, MMPs other than MMP-9 are required for the passage of the cells through unmineralized type I collagen of the nascent bone collar, and play a role in resorption of mineralized matrix. MMP-9 stimulates the solubilization of unmineralized cartilage by MMP-13, a collagenase highly expressed in hypertrophic cartilage before osteoclast invasion. Hypertrophic cartilage also expresses vascular endothelial growth factor (VEGF), which binds to extracellular matrix and is made bioavailable by MMP-9 (Bergers, G., R. Brekken, G. McMahon, T.H. Vu, T. Itoh, K. Tamaki, K. Tanzawa, P. Thorpe, S. Itohara, Z. Werb, and D. Hanahan. 2000. Nat. Cell Biol. 2:737-744). We show that VEGF is a chemoattractant for osteoclasts. Moreover, invasion of osteoclasts into the hypertrophic cartilage requires VEGF because it is inhibited by blocking VEGF function. These observations identify specific actions of MMP-9 and VEGF that are critical for early bone development.

Human breast cancer cells induced angiogenesis, recruitment, and activation of osteoclasts in osteolytic metastasis.

PURPOSE: The purpose of this study was to elucidate the potential of human breast cancer cells (BCC) to induce matrix degradation and neo-vascularization, essential for continued tumor growth, in osteolytic lesions. METHODS: BCC were inoculated into the left cardiac ventricle of female athymic mice and osteolytic lesions were radiologically visualized within 4 weeks from inoculation. RESULTS: Histomorphometric analysis of bone sections revealed a significant increase in the number and maturity of osteoclasts (OCl) lining the bone surfaces next to tumor tissue when compared to corresponding bone surfaces in healthy mice. In addition, a large number of newly formed blood vessels could be visualized by immunohistochemistry at the periphery of and within tumor tissue. When bone marrow (BM) cells were cultured in the presence of BCC the OCl formation was increased threefold. These OCl were also found to be more mature and to have greater resorptive activity. Moreover, BCC were found to stimulate proliferation, migration, and differentiation of BM-derived endothelial cells. CONCLUSIONS: Matrix destruction and neo-vascularization are accomplished by BCC arrested in the BM cavity by increasing recruitment and activity of OCl and by induction of angiogenesis within or in proximity to the tumor tissue.

Quantification of osteoclastic resorption of the bovine otic capsule in vitro by an enzyme-linked immunosorbent assay.

The bony shell surrounding the inner ear is known to have a very pronounced centripetal inhibition of remodelling in vivo, with almost no bone turnover immediately adjacent to the perilymphatic spaces and a gradually increasing turnover rate towards outer parts of the bony otic capsule. By the use of in vitro markers of bone resorption, including an enzyme-linked immunosorbent assay for quantification of type I collagen degradation and a colorimetric enzyme assay for quantification of osteoclast tartrate-resistant acid phosphatase activity, this study demonstrates that there are no ex vivo differences in bone matrix resorption between the inner and outer parts of the otic capsule when exposed to seeded osteoclasts from rabbits. Thus, the unique spatial distribution of perilabyrinthine bone turnover is not caused by a shift in resorbability from inner to outer capsular bone that is due to inherent bone quality differences particular to these bone compartments. More likely, the sustained action of some intravital 'field force', originating from the inner ear spaces, is responsible for the unique spatial distribution of the otic capsular bone turnover found in vivo. Though the character of this force is not yet defined, it is appealing to relate it to the large electromagnetic potential gradient present in the inner ear.

Proteinases in bone resorption: obvious and less obvious roles.

Bone resorption is critical for the development and the maintenance of the skeleton, and improper regulation of bone resorption leads to pathological situations. Proteinases are necessary for this process. In this review, we show that this need of proteinases is not only because they are required for the solubilization of bone matrix, but also because they are key components of the mechanism that determines where and when bone resorption will be initiated. Moreover, there are indications that proteinases may also determine whether resorption will be followed by bone formation. Some of the proteinases involved in these different steps of the resorption processes were recently identified, as for instance cathepsin K, MMP-9 (gelatinase B), and interstitial collagenase. However, there is also increasing evidence showing that the critical proteinase(s) may vary depending on the bone type or on other factors.

Solid phase combinatorial library of phosphinic peptides for discovery of matrix metalloproteinase inhibitors.

A solid phase combinatorial library of 165,000 phosphinic peptide inhibitors was prepared and screened for activity against MMP-12. The inhibitors of the library had the structure XXX-Gpsi(PO2H-CH2)L-XXX, in which X is an arbitrary amino acid and Gpsi(PO2H-CH2)L is a Gly-Leu phosphinic dipeptide analogue. The library was constructed as a one-bead-two-compounds library so that every bead contained a common quenched fluorogenic substrate and a different putative inhibitor. In addition, the inhibitor part was prepared by ladder synthesis. After incubation with MMP-12, beads containing active inhibitors were selected, and the inhibitor sequences were recorded using MALDI-TOF MS. Statistical analysis of the sequences obtained from 86 beads gave rise to a consensus sequence which was resynthesized along with 20 related sequences. Three truncated sequences and 16 sequences originally present on beads were also resynthesized. The inhibitors were investigated in an enzyme kinetic assay with MMP-12 showing that the compounds derived from the consensus sequence were strong inhibitors with Ki values down to 6 nM, whereas the sequences originally present on beads varied in potency with Ki values from micromolar to nanomolar. Truncated sequences derived from the consensus sequence were poor inhibitors of MMP-12.

Identification of breast cancer cell line-derived paracrine factors that stimulate osteoclast activity.

Metastatic breast cancer causes destruction of significant amounts of bone, and, although bone is the most likely site of breast cancer metastasis, little is understood about interactions between tumor cells and bone-resorbing osteoclasts. We have investigated the paracrine factors produced by breast cancer cells that are involved in increasing osteoclast activity. We have determined by immunoassay that the human breast cancer cell line MDA MB 231 (231) cultured in serum-free medium secretes transforming growth factors type beta(TGF-beta) 1 and 2, macrophage colony-stimulating factor (M-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL) -1 and -6, tumor necrosis factor alpha (TNF-alpha), insulin-like growth factor II (IGF II), and parathyroid hormone-related peptide. To determine which of these are involved in increased bone destruction, we have fractionated serum-free 231-conditioned media and measured these fractions for effects on osteoclast resorption activity using multiple activity assays. The pattern of responses was complex. Several fractions stimulated osteoclast resorption either by increasing the number of osteoclasts binding to the bone or by elevating the resorption activity of the individual osteoclasts. Other fractions inhibited osteoclast activity. Analysis of active fractions for the factors identified in the 231-conditioned medium revealed that the presence of TNF-alpha and IGF-II was restricted to separate fractions that stimulated osteoclast resorption activity. The fractions that inhibited osteoclast resorption activity contained M-CSF, IL-6, TGF-beta2, and GM-CSF. No TGF-beta1 or IL-1 was detected in any of the active fractions. Our data support the hypothesis that breast cancer cells modulate osteoclast activity using multiple regulatory factors that increase both the number of mature osteoclasts attached to the bone and the bone resorption activity of these individual osteoclasts. Once it is understood how metastatic breast cancer elevates osteoclast-mediated bone loss, effective therapies to slow the progression and/or prevent this bone loss will become possible.

The collagenolytic activity of cathepsin K is unique among mammalian proteinases.

Type I collagen fibers account for 90% of the organic matrix of bone. The degradation of this collagen is a major event during bone resorption, but its mechanism is unknown. A series of data obtained in biological models strongly suggests that the recently discovered cysteine proteinase cathepsin K plays a key role in bone resorption. Little is known, however, about the actual action of cathepsin K on type I collagen. Here, we show that the activity of cathepsin K alone is sufficient to dissolve completely insoluble collagen of adult human cortical bone. We found that the collagenolytic activity of cathepsin K is directed both outside the helical region of the molecule, i.e. the typical activity of cysteine proteinases, and at various sites inside the helical region, hitherto believed to resist all mammalian proteinases but the collagenases of the matrix metalloproteinase family and the neutrophil elastase. This property of cathepsin K is unique among mammalian proteinases and is reminiscent of bacterial collagenases. It is likely to be responsible for the key role of cathepsin K in bone resorption.

PEGA supports for combinatorial peptide synthesis and solid-phase enzymatic library assays.

Permeable resins cross-linked with long PEG chains were synthesized for use in solid-phase enzyme library assays. High molecular weight bis-amino-polyethylene glycol (PEG) 4000, 6000, 8000 were synthesized by a three-step reaction starting from PEG-bis-OH. Macromonomers were synthesized by partial or di-acryloylation of bis-amino-PEG derivatives. Bis/mono-acrylamido-PEG were copolymerized along with acrylamide by inverse suspension copolymerization to yield a less cross-linked resin (Type I, compounds 6-9). Furthermore, acryloyl-sarcosin ethyl ester was co-polymerized along with bis-acrylamido PEG to obtain more crosslinked capacity resin (Type II, compounds 13-19). N,N-Dimethylacrylamide was used as a co-monomer in some cases. The polymer was usually obtained in a well-defined beaded form and was easy to handle under both wet and dry conditions. The supports showed good mechanical properties and were characterized by studying the swelling properties, size distribution of beads, and by estimating the amino group capacity. Depending on the PEG chain length, the monomer composition and the degree of cross-linking the PEGA supports showed a high degree of swelling in a broad range of solvents, including water, dichloromethane, DMF, acetonitril, THF and toluene: no swelling was observed in diethyl ether. The PEGA resins (Type I) with an amino acid group capacity between 0.07 and 1.0 mmol/g could be obtained by variation of the monomer composition in the polymerization mixture. Fluorescent quenched peptide libraries were synthesized on the new polymer using a multiple column library synthesizer and incubated with the matrix metalloproteinase MMP-9 after it had been activated by 4-aminophenyl mercuric acetate resulting in 67/83 kDa active enzyme. The bright beads were separated manually under a fluorescence microscope and sequenced to obtain peptide substrates for MMP-9. After treatment with ethylene diamine, high-loaded resins (Type II) have been employed in continuous flow peptide synthesis to yield peptides in excellent yield and purity.

The migration of purified osteoclasts through collagen is inhibited by matrix metalloproteinase inhibitors.

The most obvious proteolytic event controlled by the osteoclast is bone matrix removal in the resorption compartment. Here, however, we investigated whether matrix metalloproteinase (MMP) activity of the osteoclast might be involved in its migration to its future bone resorption site. We seeded either nonpurified or purified osteoclasts onto either uncoated or collagen-coated dentine slices and cultured them in the presence or absence of specific MMP inhibitors. When nonpurified osteoclasts were cultured on uncoated dentine, MMP inhibitors did not prevent pit formation, as previously reported. However, when collagen-coated dentine was used, pit formation was strongly inhibited by MMP inhibitors. The same results were obtained when performing these experiments with purified osteoclasts, thus demonstrating the ability of osteoclasts by themselves to migrate through collagen via an MMP-dependent pathway. This demonstration was confirmed by using collagen-coated invasion chambers. In addition, the invasions were not, or only slightly, inhibited by inhibitors of serine proteinases, cysteine proteinases, and carbonic anhydrase, though the latter two are well established bone resorption inhibitors that strongly inhibited pit formation. It is concluded that osteoclasts can migrate through collagen in the absence of other cells and that this migration relies on MMP activity, whereas other enzymes typically required for bone removal in the resorption compartment are not essential for migration. Some of the osteoclast MMPs might thus be relevant to the migratory/invasive activity of the osteoclast, rather than to its bone resorptive activity itself.

Pituitary adenylyl cyclase-activating polypeptides and vasoactive intestinal peptide inhibit bone resorption by isolated rabbit osteoclasts.

Nerve fibres present in the periosteum, cortical bone and bone marrow are proposed to be involved in the regulation of bone metabolism by the release of neuropeptides acting locally on bone cells. The present study describes the effects of vasoactive intestinal polypeptide (VIP), shown to be present in the vicinity of bone, and the two related neuropeptides pituitary adenylyl cyclase-activating polypeptide(1-38) (PACAP-38) and PACAP-27 on bone resorption in vitro induced by osteoclasts isolated from 10-day-old rabbits. Bone resorption was measured as the number and total area of pits formed by tartrate-resistant acid phosphatase-positive multinucleated cells (TRAP + MNCs) cultured for 3 days on devitalized slices of bovine cortical bone. All three neuropeptides had significant inhibitory effects on both the number and area of pits formed. At a high concentration (10(-7) M) the mean +/- S.E.M. reductions in the total area resorbed compared with controls were 70.3 +/- 8.2, 45.2 +/- 7.3 and 63.4 +/- 7.2% for PACAP-38, PACAP-27 and VIP, respectively. The corresponding values for the apparent dissociation constants were 0.93 +/- 0.30, 3.2 +/- 1.6 and 0.35 +/- 0.14 nM, respectively. The number of TRAP + MNCs was unaffected by application of neuropeptides. Autoradiography showed the presence of 125I-VIP binding sites on some stromal cells, whereas osteoclasts had no binding sites for 125I-VIP. A high number of 125I-calcitonin binding sites was demonstrated on osteoclasts in the same preparation. The signal transduction pathway remains to be fully elucidated but seems to be partly dependent on changes in intracellular calcium concentrations, since a number of stromal cells responded to application of 10(-8) M PACAP-38 or VIP, and at least partly independent of cAMP accumulation. Trifluoperazine and mellitin, two selective calmodulin inhibitors, failed to block the VIP-induced inhibition of bone resorption. Our results demonstrate a non-toxic and probably stromal cell-derived effect of PACAP-38, PACAP-27 and VIP on isolated rabbit osteoclasts, resulting in a potent inhibition of bone resorption in vitro. The signal transduction pathway for inhibition induced by PACAP-38, PACAP-27 and VIP may be mediated partly by changes in intracellular Ca2+ in stromal cells.

Evidence of a correlation of estrogen receptor level and avian osteoclast estrogen responsiveness.

Isolated osteoclasts from 5-week-old chickens respond to estradiol treatment in vitro with decreased resorption activity, increased nuclear proto-oncogene expression, and decreased lysosomal enzyme secretion. This study examines osteoclasts from embryonic chickens and egg-laying hens for evidence of estrogen responsiveness. Although osteoclasts from both of these sources express estrogen receptor mRNA and protein, estradiol treatment had no effect on resorption activity. In contrast to the lack of effect on resorption, estradiol treatment for 30 minutes resulted in steady-state mRNA levels of c-fos and c-jun increasing in osteoclasts from embryonic chickens and decreasing in osteoclasts from egg-laying hens. These data suggest that a nuclear proto-oncogene response may not be involved in estradiol-mediated decreased osteoclast resorption activity. To examine the influence of circulating estrogen on osteoclast estrogen responsiveness, 5-week-old chickens were injected with estrogen for 4 days prior to sacrifice. Estradiol treatment of osteoclasts from these chickens did not decrease resorption activity in vitro. Transfection of an estrogen receptor expression vector into osteoclasts from the estradiol-injected chickens and egg-laying hens restored estrogen responsiveness. Osteoclasts from 5-week-old chickens and estradiol treated 5-week-old chickens transfected with the estrogen receptor expression vector contained significantly higher levels of estrogen receptor protein and responded to estradiol treatment by decreasing secretion of cathepsins B and L and tartrate-resistant acid phosphatase. In contrast, osteoclasts from embryonic chickens, egg-laying hens, and estradiol-treated 5-week-old chickens either untransfected or transfected with an empty expression vector did not respond similarly. These data suggest that modulation of osteoclast estrogen responsiveness may be controlled by changes in the osteoclast estrogen receptor levels.

Identification and characterization of the insulin-like growth factor I receptor in mature rabbit osteoclasts.

In this study, the insulin-like growth factor I (IGF-I) receptor was identified in rabbit osteoclasts at mRNA and protein levels by in situ hybridization and autoradiography, respectively. Using highly purified mature osteoclasts, the IGF-I receptor was characterized on the molecular level according to its size and its affinity and number per osteoclast by isolation of the receptor-ligand complex and by binding studies, respectively, and on the cellular level according to the response of mature osteoclasts to IGF-I stimulation. In situ hybridization and autoradiography experiments showed that osteoclasts express IGF-I receptor mRNA and IGF-I binding sites. Chemical cross-linking of 125I-IGF-I bound to the purified mature osteoclasts and subsequent sodium dodecyl sulfide-polyacrylamide gel electrophoresis revealed the specific binding of 125I-IGF-I in complexes with molecular masses of 130 and 230-RD consistent with binding to the IGF-I receptor. In competition experiments, 125I-IGF-I binding to mature osteoclasts was dose-dependently reduced by unlabeled IGF-I in the picomolar range, whereas 20 nM insulin did not reduce the binding of 125I-IGF-I binding. The calculated receptor number was 6000 per osteoclast, and the Kd was 0.10 nM. Searching for a role of the IGF-I receptor in mature osteoclasts, we found no significant influence of IGF-I on the levels of the proform of matrix metaloproteinase 9 and tartrate-resistant acid phosphatase. However, the induction of nuclear fragmentation in serum-depleted cultures of purified mature osteoclasts was dose-dependently inhibited by IGF-I in the picomolar range, but not by 1 nM insulin. These data show that functionally active IGF-I receptor is present in mature osteoclasts.

Identification of the membrane-type matrix metalloproteinase MT1-MMP in osteoclasts.

The osteoclasts are the cells responsible for bone resorption. Matrix metalloproteinases (MMPs) appear crucial for this process. To identify possible MMP expression in osteoclasts, we amplified osteoclast cDNA fragments having homology with MMP genes, and used them as a probe to screen a rabbit osteoclast cDNA library. We obtained a cDNA of 1,972 bp encoding a polypeptide of 582 amino acids that showed more than 92% identity to human, mouse, and rat membrane-type 1 MMP (MT1-MMP), a cell surface proteinase believed to trigger cancer cell invasion. By northern blotting, MT1-MMP was found to be highly expressed in purified osteoclasts when compared with alveolar macrophages and bone stromal cells, as well as with various tissues. In situ hybridization on bone sections showed that MT1-MMP is expressed also in osteoclasts in vivo. Antibodies recognizing MT1-MMP reacted with specific plasma membrane areas corresponding to lamellipodia and podosomes involved, respectively, in migratory and attachment activities of the osteoclasts. These observations highlight how cells might bring MT1-MMP into contact with focal points of the extracellular matrix, and are compatible with a role of MT1-MMP in migratory and attachment activities of the osteoclast.

Induction of osteocalcin gene expression in vitro by progesterone.

The effect of progesterone on osteocalcin gene expression was investigated in order to understand how progesterone acts on osteoblast in vitro. For this purpose, fetal rat calvarial osteoblasts were cultured in the medium containing 10(-9) M-10(-6) M progesterone respectively for two weeks while the cells progressing differentiation. Northern blot analysis showed that progesterone increased the level of osteocalcin mRNA in a dose-dependent manner. Among those doses of progesterone employed in this study, only 10(-6) M hormone caused a significant induction of osteocalcin gene expression (2.3 fold, P < 0.01). Our results demonstrated for the first time that the osteocalcin gene expression is regulated by progesterone. In addition, the results obtained from our study provides a good evidence for the direct effect of progesterone on osteoblast in vitro.

[Effects of progesterone on proliferation and differentiation of fetal rat calvarial osteoblasts in vitro].

OBJECTIVES: To investigate the influence of progesterone on proliferation and differentiation of osteoblast at the levels of gene expression and cell functions. METHODS: Fetal rat calvarial osteoblasts were cultured in vitro in the presence of (10(-9) mol/L-10(-6) mol/L) progesterone. Cell proliferation, alkaline phosphalase (ALP) activity, osteocalcin mRNA expression and osteocalcin secretion in the medium and bone nodule formation were analyzed. RESULTS: Progesterone did not influence cell proliferation; Progesterone enhanced the ALP activity in rat osteoblasts; Progesterone stimulated osteocalcin mRNA expression in a dose-dependent manner and increased the amount of osteocalcin in the culture medium; Progesterone increased both number and area of bone nodule formation. CONCLUSION: Progesterone has a multi-stimulating effect on the differentiation of fetal rat calvarial osteoblast, hut no effect on cell proliferation.

Interaction between effects of parathyroid hormone and bisphosphonate on regulation of osteoclast activity by the osteoblast-like cell line UMR-106.

Treatment of osteoblasts by either parathyroid hormone (PTH) or bisphosphonate can affect their regulation of the bone-resorbing activity of osteoclasts in vitro, leading to increased and decreased resorption, respectively. To address this issue, we have examined the interaction between the effects of PTH and bisphosphonate on the regulation of osteoclast activity by the PTH-responsive osteoblast-like cell line UMR-106. When rat osteoclasts were cocultured with UMR-106 cells on bovine bone slices in the presence of 10(-8) mol/L PTH, the number of resorption pits was increased 4.2-fold, whereas the addition of UMR-106 cells or PTH alone had no effect. Pretreatment of the UMR-106 cells for 5 min with increasing concentrations of either of the bisphosphonates, clodronate, and ibandronate before coculture with osteoclasts in 10(-8) mol/L PTH, caused a dose-dependent reduction in the formation of resorption pits, reaching the maximal inhibition level (60%-75% below the control) at approximately 10(-9) mol/L clodronate and 10(-11) mol/L ibandronate. Addition of conditioned medium (CM) from untreated UMR-106 cells to rat osteoclasts had no effect on pit formation, whereas CM from UMR-106 cells pretreated with ibandronate reduced the osteoclastic bone resorption by approximately 40%. However, this effect was abolished by the subsequent culture of the ibandronate-pretreated UMR-106 cells in 10(-8) mol/L PTH before harvesting the CM, because both this CM and CM from non-pretreated UMR-106 cells cultured in 10(-8) mol/L PTH caused an approximately 75% increase in pit formation when added to rat osteoclasts. In conclusion, osteoclastic bone resorption can be directly affected independently as well as at the same time by the ibandronate-induced osteoclast-inhibiting factor and the PTH-induced osteoclast-stimulating factor. The final level of bone resorption depends on the relative concentration of these two factors.