Effects of transient PTH on early proliferation, apoptosis, and subsequent differentiation of osteoblast in primary osteoblast cultures

In primary calvarial osteoblast cultures derived from transgenic mice expressing green fluorescent protein (GFP) under the control of 3.6-kb Col1a1 promoter, the emergence of GFP signal marks the transition of multipotential osteoprogenitors into preosteoblasts. Early transient treatment (days 1-7) of these cultures with parathyroid hormone (PTH) has an anabolic effect that is not associated with an increase in total DNA content or cell number in day 21 cultures. In the present study, the effect of early PTH treatment on cell proliferation and apoptosis was examined in greater detail in GFP(+) and GFP(-) cells using flow cytometry. In preconfluent cultures, PTH significantly reduced the proportion of cells in S phase but increased those in G(0)/G(1) and G(2)+M phases in both GFP(+) and GFP(-) subpopulations. PTH decreased apoptosis only in GFP(-) but not GFP(+) cells, indicating an increased survival of GFP(-) cells. In contrast, PTH did not change the amounts of cell proliferation and apoptosis seen in either compartment after these cultures reached confluence. To further assess the effect of early PTH treatment on osteogenic differentiation, secondary cultures of sorted GFP(+) or GFP(-) cells were obtained from day 7 primary cultures that had been treated for 1 wk with PTH. This treatment resulted in larger areas of GFP expression accompanied by increased xylenol orange/von Kossa staining in the secondary cultures of GFP fractions. Early transient PTH treatment appears to enhance the commitment of progenitor cells to an osteogenic fate and results in a higher proportion of cells that achieve full osteoblast differentiation.     

Mechanisms underlying catabolic and anabolic functions of parathyroid hormone on bone by combination of culture systems of mouse cells

Since bone resorption and formation by continuous and intermittent parathyroid hormone (PTH) treatments involve various types of cells in bone, this study examined the underlying mechanism by combining culture systems using mouse primary calvarial osteoblasts and bone marrow cells. The PTH/PTHrP receptor (PTH1R) expression and the cAMP accumulation in response to PTH were increased in accordance with the differentiation of osteoblasts. Osteoclast formation was strongly induced by continuous PTH treatment in the monolayer co‐culture of osteoblasts and bone marrow cells, which was associated with RANKL expression in differentiated osteoblasts. Bone formation determined by ALP activity and the type I collagen mRNA expression was stimulated by intermittent PTH treatment in the monolayer co‐culture and in the bone marrow cell layer of the separated co‐culture in a double chamber dish, but not in the culture of bone marrow cells alone. The stimulation in the separated co‐culture, accompanied by IGF‐I production by osteoblasts, was abolished when bone marrow cells were derived from knockout mice of insulin‐receptor substrate‐1 (IRS‐1?/?) or when osteoblasts were from PTH1R?/? mice. We conclude that differentiated osteoblasts are most likely the direct target of both continuous and intermittent PTH, while bone marrow cells are likely the effector cells. The osteoblasts stimulated by continuous PTH express RANKL which causes osteoclastogenesis from the precursors in bone marrow via cell‐to‐cell contact, leading to bone resorption; while the osteoblasts stimulated by intermittent PTH secrete IGF‐I which activates IRS‐1 in osteoblast precursors in bone marrow via a paracrine mechanism, leading to bone formation. J. Cell. Biochem. 109: 755–763, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Parathyroid hormone (1-34) regulates integrin expression in vivo in rat osteoblasts.

Intermittent administration of parathyroid hormone (PTH) activates new sites of bone formation by stimulating osteoblast differentiation and function resulting in an increase in bone mass. Because integrins have been shown to play a crucial role in osteoblast differentiation and bone formation, in the present study, we evaluated whether human PTH (1-34) upon administration to rats, influenced integrin expression in osteoblastic cells isolated from the metaphysis and the diaphysis of rat long bones. Initial immunohistochemical evaluation of bone sections demonstrated that the osteoblasts expressed at least alphav, alpha2, alpha3, and alpha5beta1 integrins. Immunocolocalization studies for integrins and vinculin established that alphav, alpha2, and alpha5beta1, but not alpha3 integrins were present in the focal adhesion sites of osteoblasts attached to FN coated surfaces. Osteoprogenitor cells isolated from metaphyseal (but not diaphyseal) marrow of rats injected with intermittent PTH (1-34) exhibited greater alphav and reduced alpha2 levels, with no apparent changes in alpha3, and alpha5beta1 integrin levels, as assessed by immunohistochemistry, Northern, and Western blot analyses. However, these changes were not observed on the same cells treated with PTH in vitro. These observations suggest that integrin modulation by PTH is likely to be indirect and that selective phenotypic expression of integrin subtypes is part of the cascade of events that lead to PTH (1-34) mediated osteoblast differentiation.     

The small GTPase RhoA is crucial for MC3T3‐E1 osteoblastic cell survival

Prolongation of cell survival through prevention of apoptosis is considered to be a significant factor leading to anabolic responses in bone. The current studies were carried out to determine the role of the small GTPase, RhoA, in osteoblast apoptosis, since RhoA has been found to be critical for cell survival in other tissues. We investigated the effects of inhibitors and activators of RhoA signaling on osteoblast apoptosis. In addition, we assessed the relationship of this pathway to parathyroid hormone (PTH) effects on apoptotic signaling and cell survival. RhoA is activated by geranylgeranylation, which promotes its membrane anchoring. In serum‐starved MC3T3‐E1 osteoblastic cells, inhibition of geranylgeranylation with geranylgeranyl transferase I inhibitors increased activity of caspase‐3, a component step in the apoptosis cascade, and increased cell death. Dominant negative RhoA and Y27632, an inhibitor of the RhoA effector Rho kinase, also increased caspase‐3 activity. A geranylgeranyl group donor, geranylgeraniol, antagonized the effect of the geranylgeranyl tranferase I inhibitor GGTI‐2166, but could not overcome the effect of the Rho kinase inhibitor. PTH 1‐34, a potent anti‐apoptotic agent, completely antagonized the stimulatory effects of GGTI‐2166, dominant negative RhoA, and Y27632, on caspase‐3 activity. The results suggest that RhoA signaling is essential for osteoblastic cell survival but that the survival effects of PTH 1‐34 are independent of this pathway. J. Cell. Biochem. 106: 896–902, 2009. © 2009 Wiley‐Liss, Inc.     

Preactivation of β-catenin in osteoblasts improves the osteoanabolic effect of PTH in type 1 diabetic mice

Type 1 diabetes (T1D) is correlated with osteopenia primarily due to low bone formation. Parathyroid hormone (PTH) is a known anabolic agent for bone, the anabolic effects of which are partially mediated through the Wnt/β-catenin signaling pathway. In the present study, we first determined the utility of intermittent PTH treatment in a streptozotocin-induced T1D mouse model. It was shown that the PTH-induced anabolic effects on bone mass and bone formation were attenuated in T1D mice compared with nondiabetic mice. Further, PTH treatment failed to activate β-catenin signaling in osteoblasts of T1D mice and was unable to improve osteoblast proliferation and differentiation. Next, the Col1–3.2 kb-CreERTM; β-cateninfx(ex3) mice were used to conditionally activate β-catenin in osteoblasts by injecting tamoxifen, and we addressed whether or not preactivation of β-catenin boosted the anabolic action of PTH on T1D-related bone loss. The results demonstrated that pretreatment with activation of osteoblastic β-catenin followed by PTH treatment outperformed PTH or β-catenin activation monotherapy and led to greatly improved bone structure, bone mass, and bone strength in this preclinical model of T1DM. Further analysis demonstrated that osteoblast proliferation and differentiation, as well as osteoprogenitors in the marrow, were all improved in the combination treatment group. These findings indicated a clear advantage of developing β-catenin as a target to improve the efficacy of PTH in the treatment of T1D-related osteopenia.     

Role of Smad3, acting independently of transforming growth factor‐β, in the early induction of Wnt‐β‐catenin signaling by parathyroid hormone in mouse osteoblastic cells

Parathyroid hormone (PTH) exerts an anabolic action on bone but the mechanisms are incompletely understood. We showed previously that PTH interacts with the canonical Wnt‐β‐catenin signaling pathway via the transforming growth factor (TGF)‐β signaling molecule, Smad3, to modulate osteoblast differentiation and apoptosis. Here, we examined which actions of Smad3 are TGF‐β‐independent in stimulating the osteoblast phenotype and PTH‐induced Wnt‐β‐catenin signaling. For this, the TGF‐β receptor type 1 [activin receptor‐like kinase (ALK5)] inhibitor (SB431542), and a Smad3 mutant in which the site normally phosphorylated by ALK5 is mutated from SSVS to AAVA, was used. PTH induced total β‐catenin and reduced phosphorylated β‐catenin levels at 1, 6, and 24 h in mouse osteoblastic MC3T3‐E1 cells. Transient transfection of Smad3AAVA inhibited the PTH induction of total β‐catenin and reduction of phosphorylated β‐catenin levels at 6 and 24 h, but not at 1 h, indicating that the early effects occur independently of TGF‐β receptor signaling. On the other hand, MC3T3‐E1 cell clones in which Smad3AAVA was stably expressed demonstrated elevated β‐catenin levels, although alkaline phosphatase (ALP) activity and mineralization were unaltered. In contrast, MC3T3‐E1 cell clones in which wild‐type Smad3 was stably expressed exhibited increased ALP activity and mineralization that were decreased by the ALK5 inhibitor, SB431542, although the β‐catenin levels induced in these cells were not modulated. In conclusion, the present study indicates that PTH induces osteoblast β‐catenin levels via Smad3 independently of, and dependently on, TGF‐β in the early and later induction phases, respectively. J. Cell. Biochem. 108: 285–294, 2009. © 2009 Wiley‐Liss, Inc.     

Osteoprogenitor cell frequency in rat bone marrow stromal populations: role for heterotypic cell-cell interactions in osteoblast differentiation

Glucocorticoids, notably dexamethasone (Dex), have been reported to be a requirement for osteoprogenitor cell differentiation in young adult rat bone marrow stromal cell populations. We have reinvestigated the requirement for Dex and analyzed the frequency of osteoprogenitor cells present. Stromal cells were grown as primary or first subcultures in the presence or absence of Dex and their expression of osteogenic markers (alkaline phosphatase activity, hormone responsiveness, and matrix molecules, including type I collagen, osteopontin, bone sialoprotein, and osteocalcin), as well as their functional capacity to differentiate to form a mineralized bone nodule, were assessed. Dex increased, but was not an absolute requirement for, the expression of osteogenic markers. Bone nodule formation was plating cell density dependent and occurred under all combinations of treatment with or without Dex but was maximal when Dex was present in both the primary and secondary cultures. Dex increased CFU-F by approximately 2-fold, but increased CFU-O (osteoprogenitor cells; bone nodule forming cells) by 5- to 50-fold depending on the cell density and duration of treatment. Neither CFU-F nor CFU-O expression followed a linear relationship in limiting dilution analysis until very high cell densities were reached, suggesting cooperativity of cell types within the population and a multitarget phenomenon leading to osteoprogenitor differentiation. When a large number of nonadherent bone marrow cells or their conditioned medium was added to the stromal cells, osteoprogenitors comprised approximately 1/100 of plated adherent cells and their expression followed a linear, single-hit relationship. By contrast, rat skin fibroblasts or their conditioned medium totally inhibited bone nodule formation. These data support the hypothesis that in marrow stroma, as in other bone cell populations such as those from calvaria, there are at least two classes of osteoprogenitor cells: those differentiating in the absence of added glucocorticoid and those requiring glucocorticoid to differentiate, that more than one cell type is limiting for stromal osteoprogenitor differentiation suggesting a role for heterotypic cell-cell interactions in osteogenesis in this tissue, and that Dex may be acting directly and/or indirectly through accessory cells in the bone marrow to alter osteoprogenitor cell expression.     

Constitutively active PTH/PTHrP receptor specifically expressed in osteoblasts enhances bone formation induced by bone marrow ablation

Bone is maintained by continuous bone formation by osteoblasts provided by proliferation and differentiation of osteoprogenitors. Parathyroid hormone (PTH) activates bone formation, but because of the complexity of cells in the osteoblast lineage, how these osteoprogenitors are regulated by PTH in vivo is incompletely understood. To elucidate how signals by PTH in differentiated osteoblasts regulate osteoprogenitors in vivo, we conducted bone marrow ablation using Col1a1‐constitutively active PTH/PTHrP receptor (caPPR) transgenic mice. These mice express caPPR specifically in osteoblasts by using 2.3 kb Col1a1 promoter and showed higher trabecular bone volume under steady‐state conditions. In contrast, after bone marrow ablation, stromal cells recruited from bone surface extensively proliferated in the marrow cavity in transgenic mice, compared to limited proliferation in wild‐type mice. Whereas de novo bone formation was restricted to the ablated area in wild‐type mice, the entire marrow cavity, including not only ablated area but also outside the ablated area, was filled with newly formed bone in transgenic mice. Bone mineral density was significantly increased after ablation in transgenic mice. Bone marrow cell culture in osteogenic medium revealed that alkaline phosphatase‐positive area was markedly increased in the cells obtained from transgenic mice. Furthermore, mRNA expression of Wnt‐signaling molecules such as LRP5, Wnt7b, and Wnt10b were upregulated after marrow ablation in bone marrow cells of transgenic mice. These results indicate that constitutive activation of PTH/PTHrP receptor in differentiated osteoblasts enhances bone marrow ablation‐induced recruitment, proliferation, and differentiation of osteoprogenitors. J. Cell. Physiol. 227: 408–415, 2012. © 2011 Wiley Periodicals, Inc.     

Loss of Gsα in the Postnatal Skeleton Leads to Low Bone Mass and a Blunted Response to Anabolic Parathyroid Hormone Therapy

Parathyroid hormone (PTH) is an important regulator of osteoblast function and is the only anabolic therapy currently approved for treatment of osteoporosis. The PTH receptor (PTH1R) is a G protein-coupled receptor that signals via multiple G proteins including G_sα. Mice expressing a constitutively active mutant PTH1R exhibited a dramatic increase in trabecular bone that was dependent upon expression of G_sα in the osteoblast lineage. Postnatal removal of G_sα in the osteoblast lineage (P-G_sα^OsxKO mice) yielded markedly reduced trabecular and cortical bone mass. Treatment with anabolic PTH(1–34) (80 μg/kg/day) for 4 weeks failed to increase trabecular bone volume or cortical thickness in male and female P-G_sα^OsxKO mice. Surprisingly, in both male and female mice, PTH administration significantly increased osteoblast numbers and bone formation rate in both control and P-G_sα^OsxKO mice. In mice that express a mutated PTH1R that activates adenylyl cyclase and protein kinase A (PKA) via G_sα but not phospholipase C via G_q/11 (D/D mice), PTH significantly enhanced bone formation, indicating that phospholipase C activation is not required for increased bone turnover in response to PTH. Therefore, although the anabolic effect of intermittent PTH treatment on trabecular bone volume is blunted by deletion of G_sα in osteoblasts, PTH can stimulate osteoblast differentiation and bone formation. Together these findings suggest that alternative signaling pathways beyond G_sα and G_q/11 act downstream of PTH on osteoblast differentiation.     

Osteogenic PTHs and vascular ossification—Is there a danger for osteoporotics?

Inflammation in vascular (mostly arterial) walls and heart valves triggered by the trans-endothelial influx of LDL particles and the action of subsequently modified (e.g., by oxidation) LDL particles can trigger true bone formation by valvar fibroblasts, by a subpopulation of re-differentiation-competent VSMCs (vascular smooth muscle cells) or by vascular pericytes. Vascular ossification can lead to heart failure and death. Elderly osteoporotic women who need osteogenic drugs to restore their lost skeletal bone are paradoxically prone to vascular ossification-the "calcification paradox." The recent introduction into the clinic of a potently osteogenic parathyroid hormone peptide, Lilly's rhPTH-(1-34)OH (Forteotrade mark), to reverse skeletal bone loss raises the question of whether this and other potently osteogenic PTHs still in clinical trial might also stimulate vascular ossification in such osteoporotic women. Indeed the VSMCs in human and rat atherosclerotic lesions hyperexpress PTHrP and the PTHR1 (or PTH1R) receptor as do maturing osteoblasts. And the evidence indicates that endogenous PTHrP with its NLS (nuclear/nucleolar localization sequence) does stimulate VSMC proliferation (a prime prerequisite for atheroma formation and ossification) via intranuclear targets that inactivate pRb, the inhibitory G1/S checkpoint regulator, by stimulating its hyperphosphorylation. But neither externally added full-length PTHrP nor the NLS-lacking PTHrP-(1-34)OH gets into the VSMC nucleus and instead they inhibit proliferation and calcification by only activating the cell's PTHR1 receptors. No PTH has an NLS and, as expected from the observations on the externally added PTHrPs, hPTH-(1-34)OH inhibits calcification by VSMCs and cannot stimulate vascular ossification in a diabetic mouse model. Encouraging though this may be for osteoporotics with their "calcification paradox," more work is needed to be sure that the skeletally osteogenic PTHs do not promote vascular ossification with its cardiovascular consequences.     

Parathyroid hormone (PTH) and hematopoiesis: new support for some old observations

Forty-seven years ago, the parathyroid hormone (PTH) in one injection of Lilly's old bovine parathyroid extract, PTE, was found to greatly increase the 30-day survival of heavily X-irradiated rats when given from 18 h before to as long as 3 h after irradiation but no later. This was the first indication that PTH might stimulate hematopoiesis. Recent studies have confirmed the relation between PTH and hematopoiesis by showing that hPTH-(1-34)OH increases the size of the hematopoietic stem cell pool in mice. The peptide operates through a cyclic AMP-mediated burst of Jagged 1 production in osteoblastic cells lining the stem cells' niches on trabecular bone surfaces. The osteoblastic cells' Jagged 1 increases the hematopoietic stem cell pool by activating Notch receptors on attached stem cells. PTH-triggered cyclic AMP signals also directly stimulate the proliferation of the hematopoietic stem cells. However, the single PTH injection in the early experiments using PTE probably increased the survival of irradiated rats mainly by preventing the damaged hematopoietic progenitors from irreversibly initiating self-destructive apoptogenesis during the first 5 h after irradiation. It has also been shown that several daily injections of hPTH-(1-34)OH enable lethally irradiated mice to survive by stimulating the growth of transplanted normal bone marrow cells. If the osteogenic PTHs currently entering or on the verge of entering the market for treating osteoporosis can also drive hematopoiesis in humans as well as rodents, they could be potent tools for reducing the damage inflicted on bone marrow by cytotoxic cancer chemotherapeutic drugs and ionizing radiation.     

Molecular cloning and functional characterization of the canine parathyroid hormone/parathyroid hormone related peptide receptor (PTH1)

Parathyroid hormone (PTH) is a major mediator of calcium and phosphate metabolism through its interactions with receptors in kidney and bone. PTH binds with high affinity to PTH1 and PTH2, members of the superfamily of G protein-coupled receptors. In order to clone the canine PTH1 receptor, a canine kidney cDNA library was screened using the human PTH1 receptor cDNA and two clones were further characterized. The longest clone was 2177 bp and contained a single open reading frame of 1785 bp, potentially encoding a protein of 595 amino acids with a predicted molecular weight of 66.4 kD. This open reading frame exhibits >91% identity to the human PTH1 receptor cDNA and >95% identity when the putative canine and human protein sequences are compared. Competition binding following transfection of the canine PTH1 receptor into CHO cells demonstrated specific displacement of ¹²⁵I-human PTH 1-34 by canine PTH 1-34, human PTH 1-34, and canine/human parathyroid hormone related peptide (PTHrP) 1-34. Treatment of canine PTH1 receptor transfected cells, but not mock transfected cells, with these ligands also resulted in increased levels of intracellular cAMP. In contrast, the non-related aldosterone secretion inhibiting factor 1-35 neither bound nor activated the canine PTH1 receptor. Northern blot analysis revealed high levels of PTH1 receptor mRNA in the kidney, with much lower, but detectable, levels in aorta, heart, lung, prostate, testis, and skeletal muscle. Together, these data indicate that we have cloned the canine PTH1 receptor and that it is very similar, both in sequence and in functional characteristics, to the other known PTH1 receptors.     

Positive and negative immunoselection for enrichment of two classes of osteoprogenitor cells.

The number of identifiable stages and expression of differentiation markers in cells of the osteoblast lineage are not well understood. In the present study, a mAb, designated rat bone marrow (RBM) 211.13, was prepared that stained selectively the osteogenic and preosteoblastic cells along the surfaces of bone in calvariae, femurs, and metatarsals. The staining was cell surface associated and coincided with that for alkaline phosphatase (APase) detected histochemically. Only cells positive for APase activity by biochemical assay and not those without APase activity (e.g., fetal rat skin) stained with RBM 211.13. By immunoblotting, RBM 211.13 recognized a band coinciding with APase activity on nonreducing/nondenaturing gels, and RBM 211.13 precipitated a protein which on reduced gels migrated with an apparent molecular mass of approximately 80 kD. RBM 211.13 labeling was abolished by phosphatidylinosital-specific phospholipase C, known to release APase from the cell surface. All of these data support the concept that RBM 211.13 recognizes the bone isoenzyme of APase. RBM 211.13 was used to sort by flow cytometry the APase-positive and APase-negative cells from mixed fetal rat calvaria (RC) cell populations. The osteoprogenitors we identified earlier that form bone nodules in vitro (Bellows, C. G., J. E. Aubin, J. N. M. Heersche, and M. E. Antosz. 1986. Calcif. Tissue Int. 36:143-154; Bellows, C. J., J. N. M. Heersche, and J. E. Aubin. 1990. Dev. Biol. 140:132-138) were found within the APase-positive pool. By immunopanning, RC cells were separated into APase-enriched (APase-positive, adherent) and APase-depleted (APase-negative, nonadherent) populations. The APase-positive fraction was enriched two-to-threefold for bone-forming osteoprogenitors compared to unfractionated cells, while the APase-negative population formed very few nodules under the same conditions. Both populations responded to the glucocorticoid dexamethasone (DEX) with an increase in bone nodule formation. However, the fold stimulation in bone formation in the APase-negative population was approximately 30-fold, while the fold stimulation in the APase-positive population was only approximately 5-fold. These data suggest that APase expression can be used for immunoselection to fractionate osteoblastic populations into an APase-positive population and a population initially APase-negative, that virtually all osteoprogenitors forming bone in vitro in the absence of added glucocorticoids reside in the APase-positive pool, and that the only osteoprogenitors present in the APase-negative pool are those requiring DEX to differentiate.