Effects of continuous and pulsatile PTH treatments on rat bone marrow stromal cells

Bone marrow stromal cells (MSCs) differentiation and proliferation are controlled by numerous growth factors and hormones. Continuous parathyroid hormone (PTH) treatment has been shown to decrease osteoblast differentiation, whereas pulsatile PTH increases osteoblast differentiation. However, the effects of PTH treatments on MSCs have not been investigated. This study showed continuous PTH treatment in the presence of dexamethasone (DEX) promoted osteogenic differentiation of rat MSCs in vitro, as demonstrated by increased alkaline phosphatase (ALP) activity, number of ALP expressing cells, and up-regulation of PTH receptor-1, ALP, and osteocalcin mRNA expressions. In contrast, pulsatile PTH treatment was found to suppress osteogenesis of rat MSCs, possibly by promoting the maintenance of undifferentiated cells. Additionally, the observed effects of PTH were strongly dependent on the presence of DEX. MSC proliferation however was not influenced by PTH independent of treatment regimen and presence or absence of DEX. Furthermore, our work raised the possibility that PTH treatment may modulate stem/progenitor cell activity within MSC cultures.     

甲状旁腺素、胰岛素样生长因子-Ⅰ对骨特征性标志蛋白的影响

为了阐明甲状旁腺素（ｐａｒａｔｈｙｒｏｉｄｈｏｒｍｏｎｅ,ＰＴＨ）、胰岛素样生长因子－Ⅰ（ｉｎｓｕｌｉｎ－ｌｉｋｅｇｒｏｗｔｈｆａｃｔｏｒ－Ⅰ,ＩＧＦ－Ⅰ）在促成骨样细胞ＲＯＳ１７／２．８增殖的同时对其分化状况的影响,研究了ＰＴＨ、ＩＧＦ－Ⅰ对骨特征性标志蛋白碱性磷酸酶（ａｌｋａｌｉｎｅｐｈｏｓｐｈａｔａｓｅ,ＡＬＰ）活性、骨钙素（ｏｓｔｅｏｃａｌ－ｃｉｎ）ｍＲＮＡ表达及骨胶原蛋白（ｃｏｌａｇｅｎ）合成的影响．结果表明,ＰＴＨ、ＩＧＦ－Ⅰ均可抑制ＡＬＰ活性,ＰＴＨ的抑制作用强于ＩＧＦ－Ⅰ,抑制作用具有时间剂量依赖性;ＰＴＨ、ＩＧＦ－Ⅰ均可诱导骨钙素ｍＲＮＡ表达增加;ＰＴＨ能够促进３Ｈ－脯氨酸参入新生小鼠颅骨组织,既ＰＴＨ能够骨胶原蛋白合成增加．这些结果提示,ＰＴＨ、ＩＧＦ－Ⅰ在促成骨样细胞ＲＯＳ１７／２．８增殖的同时也促进其分化,表明在成骨细胞的生发成熟过程中,增殖、分化现象相辅相成,相伴而行．     

Bone microenvironment-related growth factors, zoledronic acid and dexamethasone differentially modulate PTHrP expression in PC-3 prostate cancer cells.

Bone metastasis microenvironment-related growth factors such as insulin-like growth factor 1 (IGF-1), transforming growth factor beta 1 (TGF-beta1), basic fibroblast growth factor (bFGF) and interleukin 6 (IL-6) show survival factor activity, thereby inhibiting chemotherapy-induced apoptosis of PC-3 prostate cancer cells in vitro. Recently, zoledronic acid has been shown to induce apoptosis in PC-3 prostate cancer cells while overexpression of parathyroid hormone-related protein (PTHrP) inhibits serum deprivation-induced apoptosis in PC-3 cells. Consequently, we have investigated whether IGF-1, TGF-beta1, bFGF, IL-6, zoledronic acid and/or dexamethasone affect the expression of the PTHrP and type I PTH/PTHrP receptor (PTH.1R) in PC-3 prostate cancer cells using relative quantitative PCR and real-time PCR (expression at mRNA level) and immunocytochemical and immunofluorescence analysis (expression at protein level). Our data show that IGF-1, TGF-beta1, bFGF and IL-6 increase PTHrP mRNA expression and its perinuclear localization, while zoledronic acid (50 muM, 100 muM for 24 h and 48 h) and dexamethasone suppress PTHrP expression in PC-3 cells. We did not detect any appreciable change of the PTH.1R expression due to IGF-1, TGF- beta1, bFGF, IL-6, zoledronic acid or dexamethasone in PC-3 cells. Therefore, it is conceivable that bone metastasis microenvironment-related survival factor/anti-apoptotic activity and zoledronic acid anticancer action/pro-apoptotic activity on PC-3 cells is mediated, at least in part, by differential modulation of PTHrP expression.     

甲状旁腺素对成骨样细胞增殖的调节作用

甲状旁腺素（ＰＴＨ）是调节钙磷代谢的经典激素,有报道ＰＴＨ对其靶细胞－成骨细胞有促增殖分化作用。经多层次、多水平的实验研究证实,ＰＴＨ对成骨样细胞ＲＯＳ１７／２．８确有促增殖作用。（１）细胞计数、ＭＴＴ［３－（４,５－ｄｉｍｅｔｈｙｌｔｈｉａ－ｚｏｌ－ｚ－ｙｉ）２,５－ｄｉｐｈｅｎｙｌｔｅｔｒａｚｏｌｉｕｍｂｒｏｍｉｄｅ］测定及ＳＲＢ（ｓｏｄｉｕｍｒｈｏｄａｍｉｎｅＢ,ＳＲＢ）染色均显示经ＰＴＨ（１０－９ｍｏｌ／Ｌ）处理的细胞,其数目明显增加;（２）３Ｈ－ＴｄＲ参入增加;（３）与增殖相关的原癌基因（ｃ－ｆｏｓ、ｃ－ｊｕｎ、ｃ－ｋｉ－ｒａｓ和ｃ－ｍｙｃ）的表达增强;（４）成骨细胞特征性蛋白－碱性磷酸酶活性降低．这些结果不仅表明该激素具有非经典样作用,同时意味着激素也参与其靶细胞增殖分化的调节作用     

Studies on the mechanisms of the skeletal anabolic action of endogenous and exogenous parathyroid hormone

Parathyroid hormone (PTH) has been viewed as catabolic for bone. Nevertheless, exogenous PTH is anabolic when administered intermittently, at a frequency that permits complete clearance between doses. In the fetus and neonate, endogenous PTH is required for normal trabecular bone formation. In older animals PTH produces net bone loss in fulfilling its calcium homeostatic role, whereas PTH-related peptide (PTHrP), acting in a paracrine/autocrine mode, is anabolic. The proliferative, differentiating, and anti-apoptotic effects of PTH on cells of the osteoblast lineage leading to anabolism can be direct, or indirect via release of local growth factors. The anabolic effect of PTH is also influenced by osteoclastic activity such that suppression of osteoclasts with anti-resorptive agents, concomitant to administering PTH, may enhance the anabolic effect by delaying a reactive osteoclastic response. In contrast, prolonged suppression of osteoclast activity prior to administering PTH appears to diminish molecular signals that increase the osteoblast pool and thereby reduces the anabolic efficacy of PTH. These observations may define the proper timing of the use of PTH as a therapeutic in diseases of bone loss. Finally, the capacity of exogenous PTH to modulate extra-osseous factors such as 1,25 dihydroxyvitamin D may also modulate its potency as an anabolic agent.     

Full-length chicken parathyroid hormone. Biosynthesis in Escherichia coli and analysis of biologic activity.

Chicken parathyroid hormone (cPTH) has been reported to stimulate adrenal steroidogenesis and to have unusual potency on traditional PTH target tissues. To evaluate these properties, chicken PTH-(1-88) has been expressed in Escherichia coli using a plasmid encoding a fusion protein which links together growth hormone, a factor Xa recognition site, and chicken PTH-(1-88). The growth hormone-cPTH fusion protein required the presence of 0.02% sodium dodecyl sulfate to remain in solution and be cleaved by factor Xa. The high performance liquid chromatography-purified recombinant cPTH-(1-88) and chemically synthesized cPTH-(1-34) had similar potency in rat osteosarcoma (ROS 17/2.8) cells, opossum kidney (OK) cells, and dispersed primary chicken kidney cells. The biologic potencies of cPTH-(1-34) and cPTH-(1-88) in radioreceptor binding and cAMP generation in both bone- and kidney-derived cell lines were less than those of human (h)PTH-(1-34). In dispersed chicken kidney cells, cAMP production by cPTH-(1-34) and cPTH-(1-88) was similar to that stimulated by human PTH-(1-34). No stimulation of steroidogenesis could be detected when recombinant chicken PTH-(1-88) was added to dispersed chicken adrenal cells. The biologic activity of recombinant chicken PTH-(1-88) purified from E. coli was comparable with that of chicken PTH-(1-88) expressed by mammalian COS cells. Thus, the full-length chicken PTH did not exhibit enhanced potency, when compared with human PTH in ROS 17/2.8, OK cell lines, and dispersed chicken kidney cells and did not demonstrate the novel steroidogenic action previously reported in adrenal cells. The successful production of chicken PTH-(1-88) will enhance our understanding of the structure-activity relationships for PTH, particularly the sequence-dependent metabolism of the hormone.     

PTH stimulates the proliferation of TE-85 human osteosarcoma cells by a mechanism not involving either increased cAMP or increased secretion of IGF-I,IGF-II or TGFβ

Injections of parathyroid hormone (PTH) result in increased bone formation in several species. Work in our laboratory and others has shown a stimulation of bone cell proliferation and growth factor production by PTH. Our purpose was to study the effects of PTH on a human bone cell line using TE-85 human osteosarcoma cells as a model. After 24 h treatment, PTH caused an increase in cell proliferation as measured by cell counts and [³H]-thymidine incorporation. Proliferation was not inhibited by an anti-transforming growth factor beta (TGFβ) antibody which could abolish stimulation by exogenous TGFβ. PTH did not stimulate cAMP production, alkaline phosphatase activity or production of insulin-like growth factors I or II (IGF-I or IGF-II) in TE-85 cells. Although basal TE-85 proliferation was slowed by incubation with the calcium channel blocking agent verapamil, PTH still caused an increase in growth rate. We conclude that PTH directly stimulates TE-85 proliferation via a mechanism not involving increased adenylate cyclase activity or increased secretion of IGF-I, IGF-II or TGFβ and may stimulate bone formation in vivo by activating some other mitogenic signal to increase bone cell proliferation.     

Impact of parathyroid hormone on bone marrow-derived stem cell mobilization and migration

Parathyroid hormone (PTH) is well-known as the principal regulator of calcium homeostasis in the human body and controls bone metabolism via actions on the survival and activation of osteoblasts. The intermittent administration of PTH has been shown to stimulate bone production in mice and men and therefore PTH administration has been recently approved for the treatment of osteoporosis. Besides to its physiological role in bone remodelling PTH has been demonstrated to influence and expand the bone marrow stem cell niche where hematopoietic stem cells, capable of both self-renewal and differentiation, reside. Moreover, intermittent PTH treatment is capable to induce mobilization of progenitor cells from the bone marrow into the bloodstream. This novel function of PTH on modulating the activity of the stem cell niche in the bone marrow as well as on mobilization and regeneration of bone marrow-derived stem cells offers new therapeutic options in bone marrow and stem cell transplantation as well as in the field of ischemic disorders.     

The int-2 gene product acts as a growth factor and substitutes for basic fibroblast growth factor in promoting the differentiation of a normal mouse mammary epithelial cell line.

We have investigated the effect of basic fibroblast growth factor (bFGF) and the related int-2 gene on the growth, transformation, and differentiation of HC11 mouse mammary epithelial cells. We show that in HC11 cells infected with int-2 retroviral expression vectors, the int-2 protein can function as a bFGF-like growth factor in stimulating: (a) HC11 cell proliferation in monolayer, (b) anchorage-independent growth in soft agar, and (c) soft agar growth of the bFGF-responsive SW13 tumor cell line. These effects are observed irrespective of whether the int-2 protein is expressed in its wild-type form or is linked to a signal peptide. A candidate bFGF receptor, which is the product of the flg gene and which may recognize the int-2 protein, is expressed at high levels in HC11 cells. Following epidermal growth factor or bFGF priming and subsequent treatment with lactogenic hormones, all of the int-2 infected and the parental HC11 cells synthesize similar levels of beta-casein. However, the autocrine expression of int-2 in HC11 cells abrogates their requirement for either exogenous epidermal growth factor or bFGF priming. These data suggest that, in HC11 cells, the growth factor activity of the int-2 gene is indistinguishable from that of bFGF and does not interfere with the mammary cell differentiation program associated with lactogenesis.     

Parathyroid hormone inhibits phosphorylation of mitogen‐activated protein kinase (MAPK) ERK1/2 through inhibition of c‐Raf and activation of MKP‐1 in osteoblastic cells

Parathyroid hormone (PTH) regulation of mitogen‐activated protein kinases (MAPK) ERK1/2 contributes to PTH regulation of osteoblast growth and apoptosis. We investigated the mechanisms by which PTH inhibits ERK1/2 activity in osteoblastic UMR 106‐01 cells. Treatment with PTH significantly inhibited phosphorylated ERK1/2 between 5 and 60 min. Transient transfection of cells with a cDNA encoding MAPK phosphatase‐1 (MKP‐1) resulted in 30–40% inhibition of pERK1/2; however MKP‐1 protein levels were only significantly stimulated by PTH after 30 mins, suggesting another mechanism for the early phase of pERK1/2 inhibition. The active upstream kinase c‐Raf phosphorylation at serine 338 (ser³³⁸) was significantly inhibited by PTH treatment within 5 min and transfection of the cells with constitutively‐active c‐Raf blocked PTH inhibition of pERK1/2. Inhibition of pERK1/2 and phosphor‐c‐Raf were seen when cells were treated with PTH(1‐34) or PTH(1‐31) analogues that stimulate cAMP, but not with PTH(3‐34), PTH(7‐34) or PTH(18‐48) that do not stimulate cAMP. Stimulation of the cells with forskolin or 8BrcAMP also inhibited pERK1/2 and c‐Raf.p338. Our results suggest that rapid PTH inhibition of ERK1/2 activity is mediated by PKA dependent inhibition of c‐Raf activity and that stimulation of MKP‐1 may contribute to maintaining pERK1/2 inhibition over prolonged time. Copyright © 2009 John Wiley & Sons, Ltd.     

Stimulation of creatine kinase BB activity by parathyroid hormone and by prostaglandin E2 in cultured bone cells.

Bone cells in culture responded to parathyroid hormone (PTH) and prostaglandin E2 (PGE2) by a 2-fold increase in creatine kinase (CK) activity. Combined treatment resulted in a higher response than with PTH alone. Calcitonin (CT) failed to stimulate CK activity, did not affect the response of CK to PTH, but inhibited slightly the increase in CK activity by PGE2. Bone-cell cultures grown in low [Ca2+] (0.125 mM), enriched in PTH-responsive osteoblast-like cells, responded to PTH, but not to PGE2 or CT, by increased CK activity. In both normal and low-[Ca2+] cultures, 8-bromo cyclic AMP did not affect CK activity, nor did it change the response of the cells to PTH, PGE2 or CT. The increase in CK activity was time- and dose-dependent and inhibited both by cycloheximide and by actinomycin D. The isoenzyme of CK stimulated was the CKBB form, the isoenzyme induced by other hormones. This appears to be the first report of the stimulation of CK activity by a polypeptide hormone or a prostaglandin. We suggest that stimulation of CKBB can serve as a marker for the action of a variety of hormones and growth promoters.     

甲状旁腺素和胰岛素样生长因子促成骨样细胞ROS 17/2.8增殖分化中的协同作用

 间歇性小剂量地给予甲状旁腺素 (parathyroid hormone,PTH)可促进成骨 .胰岛素样生长因子 - I(insulin- like growth factor- I,IGF- I)由成骨细胞所产生并贮存于骨基质中 ,可促进成骨细胞的增殖分化 .为进一步了解向钙性激素和骨源性生长因子对骨生长的影响 ,利用成骨样细胞 ROS1 7/ 2 .8进行体外实验 ,观察了 PTH和 IGF- I这两种在骨生长和代谢中有重要作用的激素和因子相互作用的效果 ,并对其相互作用机制作出初步探讨 .结果显示 :联合使用 IGF- I及 PTH(间歇性给药 )时 ,(1 ) SRB(sodium rhodamine B,SRB)染色显示经 PTH(1 0 -9mol/ L,间歇给药 )和 IGF- I(1 0 -9mol/ L)联合处理的细胞 ,其数目明显增加 ,且明显高于单独处理组 ;(2 ) 3H- Td R参入增加 ,也明显高于单独处理组 ;(3)与增殖相关的原癌基因 (c- fos,c- jun,c- ki- ras)的表达增强 ,明显高于单独处理组 ;(4)骨钙素 (osteocalcin)基因 m RNA表达增强 ,明显高于单独处理组 ;(5) IGF- I(1 0 -8mol/L,1 0 -9mol/ L)可使 PTH受体基因 m RNA表达增强 .这些结果提示 PTH和 IGF- I在成骨样细胞ROS 1 7/ 2 .8增殖分化中具有协同作用 ,原癌基因的表达增强可能是其作用的一个环节 .此外 ,IGF- I可能通过增强 PTH受体表达 ,使细胞对 PTH的反应性增强     

Stimulation of extracellular signal-regulated kinases and proliferation in rat osteoblastic cells by parathyroid hormone is protein kinase C-dependent

Parathyroid hormone (PTH) is known to have both catabolic and anabolic effects on bone. The dual functionality of PTH may stem from its ability to activate two signal transduction mechanisms: adenylate cyclase and phospholipase C. Here, we demonstrate that continuous treatment of UMR 106-01 and primary osteoblasts with PTH peptides, which selectively activate protein kinase C, results in significant increases in DNA synthesis. Given that ERKs are involved in cellular proliferation, we examined the regulation of ERKs in UMR 106-01 and primary rat osteoblasts following PTH treatment. We demonstrate that treatment of osteoblastic cells with very low concentrations of PTH (10(-12) to 10(-11) m) is sufficient for substantial increases in ERK activity. Treatment with PTH-(1-34) (10(-8) m), PTH-(1-31), or 8-bromo-cAMP failed to stimulate ERKs, whereas treatment with phorbol 12-myristate 13-acetate, serum, or PTH peptides lacking the N-terminal amino acids stimulated activity. Furthermore, the activation of ERKs was prevented by pretreatment of osteoblastic cells with inhibitors of protein kinase C (GF 109203X) and MEK (PD 98059). Treatment of UMR cells with epidermal growth factor (EGF), but not PTH, promoted tyrosine phosphorylation of the EGF receptor. Transient transfection of UMR cells with p21(N17Ras) did not block activation of ERKs following treatment with low concentrations of PTH. Thus, activation of ERKs and proliferation by PTH is protein kinase C-dependent, but stimulation occurs independently of the EGF receptor and Ras activation.     

Epidermal growth factor receptor, platelet-derived growth factor receptor, and c-erbB-2 receptor activation all promote growth but have distinctive effects upon mouse mammary epithelial cell differentiation.

Three different receptor tyrosine kinases, epidermal growth factor (EGF), c-erbB-2/neu, and platelet-derived growth factor (PDGF) receptors, have been found to be present in the mouse mammary epithelial cell line HC11. We have investigated the consequences of receptor activation on the growth and differentiation of HC11 cells. HC11 cells are normal epithelial cells which maintain differentiation-specific functions. Treatment of the cells with the lactogenic hormones glucocorticoids and prolactin leads to the expression of the milk protein beta-casein. Activation of EGF receptor has a positive effect on cell growth and causes the cells to become competent for the lactogenic hormone response. HC11 cells respond optimally to the lactogenic hormone mixture and synthesize high levels of beta-casein only if they have been kept previously in a medium containing EGF. Transfection of HC11 cells with the activated rat neuT receptor results in the acquisition of competence to respond to the lactogenic hormones even if the cells are grown in the absence of EGF. The activation of PDGF receptor, through PDGF-BB, also stimulates the growth of HC11 cells. Cells kept only in PDGF do not become competent for lactogenic hormone induction. The results show that activation of the structurally related EGF and c-erbB-2/neu receptors, but not the PDGF receptor, allows the HC11 cells to subsequently respond optimally to lactogenic hormones.     

Paracrine interactions in bone-secreted products of osteoblasts permit osteoclasts to respond to parathyroid hormone

During bone remodeling, activation of resorption is followed by a cycle of formation and this ordered sequence of events has long suggested that local interactions between osteoclasts and osteoblasts are an important regulatory mechanism in bone metabolism. To study this phenomenon, we have prepared bone cells containing primarily osteoclasts by brief digestion of mice calvariae in collagenase, overnight attachment to polystyrene tissue culture flasks in serumless medium supplemented with OB (osteoblast) cell conditioned medium and subsequent growth in low serum. These OC (osteoclast) cells were found to be highly enriched in acid phosphatase activity and expressed cAMP responses to PTH (parathyroid hormone) and prostaglandin E2 but exhibited no PTH-stimulated hyaluronate synthesis in contrast to prostaglandin E2. PTH effects on hyaluronate, however, could be restored upon coculture of OC cells with OB cells (noncontact) or with OB cell conditioned medium, thereby suggesting that OB cells regulate OC cell PTH responsiveness and/or differentiation by soluble cell products secreted into the medium.