Rebecca R. Miles

Catabolic Effects of Continuous Human PTH (1–38) in Vivo Is Associated with Sustained Stimulation of RANKL and Inhibition of Osteoprotegerin and Gene-Associated Bone Formation

Continuous infusion of PTH in vivo results in active bone resorption. To investigate the molecular basis of the catabolic effect of PTH in vivo, we evaluated the role of OPG and RANKL, which are known to influence osteoclast formation and function. Weanling rats fed a calcium-free diet were parathyroidectomized and infused with PTH via an Alzet pump to examine: 1) the changes of serum-ionized calcium and osteoclast number, 2) the expression of OPG/RANKL mRNA and protein, and 3) the expression of osteoblast phenotype bone formation-associated genes such as osteoblast specific transcription factor, osteocalcin, bone sialoprotein, and type I collagen. PTH (1–38) (0.01–20 μg/100 g) continuous infusion for 1–24 h resulted in a dose-dependent increase in serum-ionized calcium in parathyroidectomized rats and a corresponding dose-dependent increase in osteoclast number, indicating an increased bone resorption. At 20 μg/100 g PTH dose level, serum-ionized calcium was 2.1-fold of the vehicle control and not differ...

Effects of parathyroid hormone on Wnt signaling pathway in bone

The Wnt signaling pathway has recently been demonstrated to play an important role in bone cell function. In previous studies using DNA microarray analyses, we observed a change in some of the molecular components of the canonical Wnt pathway namely, frizzled‐1 (FZD‐1) and axil, in response to continuous parathyroid hormone (PTH) treatment in rats. In the present study, we further explored other components of the Wnt signaling pathway in rat distal metaphyseal bone in vivo, and rat osteoblastic osteosarcoma cells (UMR 106) in culture. Several Wnt pathway components, including low‐density lipoprotein‐receptor‐related protein 5 (LRP5), LRP6, FZD‐1, Dickkopf‐1 (Dkk‐1), and Kremen‐1 (KRM‐1), were expressed in bone in vivo and in osteoblasts in vitro. Continuous exposure to PTH (1–38) both in vivo and in vitro upregulated the mRNA expression of LRP6 and FZD‐1 and decreased LRP5 and Dkk‐1. These effects in UMR 106 cells were associated with an increase in β‐catenin as measured by Western blots and resulted in functional activation (three to six‐fold) of a downstream Wnt responsive TBE6‐luciferase (TCF/LEF‐binding element) reporter gene. Activation of the TBE6‐luciferase reporter gene by PTH (1–38) in UMR 106 cells was inhibited by the protein kinase A (PKA) inhibitor, H89. Activation was mimicked by PTH (1–31), PTH‐related protein (1–34), and forskolin, but both PTH (3–34) and (7–34) had no effect. These findings suggest that the effect of PTH on the canonical Wnt signaling pathway occurs at least in part via the cAMP‐PKA pathway through the differential regulation of the receptor complex proteins (FZD‐1/LRP5 or LRP6) and the antagonist (Dkk‐1). Taken together, these results reveal a possible role for the Wnt signaling pathway in PTH actions in bone.

The osteoblast-specific transcription factor Cbfa1 contributes to the expression of osteoprotegerin, a potent inhibitor of osteoclast differentiation and function.

Bone formation and resorption are tightly coupled under normal conditions, and the interaction of osteoclast precursors with cells of the osteoblast lineage is a prerequisite for osteoclast formation. Cbfa1 is an osteoblast-specific transcription factor that is essential for osteoblast differentiation and bone formation. At present, it is not known whether Cbfa1 regulates any of the osteoblast-derived factors involved in the bone resorption pathway. Osteoprotegerin (OPG) is an osteoblast-secreted glycoprotein that functions as a potent inhibitor of osteoclast differentiation and bone resorption. Cloning and computer analysis of a 5.9-kilobase human OPG promoter sequence revealed the presence of 12 putative Cbfa1 binding elements (osteoblast-specific element 2 (OSE2)), suggesting a possible regulation of OPG by Cbfa1. We cloned the promoter upstream of the β-galactosidase reporter gene (pOPG5.9βgal) and evaluated whether Cbfa1 could regulate its expression in transient transfection assays. The 5.9-kilobase promoter directed increased levels of reporter gene expression, reminiscent of OPG protein levels in osteoblastic cell lines (BALC and U2OS) as compared with the nonosteoblastic cell line COS1. Cotransfection of a Cbfa1 expression construct along with pOPG5.9βgal reporter construct led to 39-, 7-, and 16-fold increases in β-galactosidase activity in COS1, BALC, and U2OS cells, respectively. Removal of all the putative OSE2 elements led to an almost complete loss of transactivation. Mutational analysis demonstrated that the proximal OSE2 element contributes to a majority of the effects of Cbfa1, and Cbfa1 bound to the proximal element in a sequence-specific manner. Further, overexpression of Cbfa1 led to a 54% increase in OPG protein levels in U2OS cells. These results indicate that Cbfa1 regulates the expression of OPG, thereby further contributing to a molecular link between bone formation and resorption.

Activation of osteocalcin transcription involves interaction of protein kinase A- and protein kinase C-dependent pathways.

Osteocalcin is a major noncollagenous protein component of bone extracellular matrix, synthesized and secreted exclusively by osteoblastic cells in the late stage of maturation, and is considered indicator of osteoblast differentiation. Osteocalcin expression is modulated by parathyroid hormone (PTH) and a variety of other factors. The cAMP-dependent protein kinase pathway has been shown previously to have an essential role in PTH signaling and regulation of osteocalcin expression. To determine the extent to which other pathways may also participate in osteocalcin expression, we used rat and human osteoblast-like cell lines to generate stably transfected clones in which the osteocalcin promoter was fused to a luciferase reporter gene. These clones were examined for their responsiveness to agents known to activate or interfere with protein kinase A (PKA)- and protein kinase C (PKC)-dependent pathways. We have found that forskolin, cAMP, and PTH, as well as insulin-like growth factor I (IGF-I) and basic fibroblast growth factor, all were effective in activating the osteocalcin promoter. Phorbol 12-myristate 13-acetate (PMA) was also a strong inducer of the promoter, indicating that PKC plays a role in expression of osteocalcin. In combination with PTH or forskolin, the effect of PMA was additive to synergistic. Calphostin C, a selective inhibitor of PKC, decreased the PMA-, PTH-, and IGF-I-induced luciferase activity in a dose-dependent manner; a PKA inhibitor, H-89, also blocked the induction by PTH and IGF-I but not by PMA. We conclude that regulation of osteocalcin transcription is mediated by both PKA-dependent and PKC-dependent mechanisms and that the respective kinases reside on a linear or convergent pathway.

Inhibition of estrogen-stimulated growth of uterine leiomyomas by selective estrogen receptor modulators

Uterine leiomyoma is the most frequent gynecologic neoplasm in women. By using a panel of cell lines derived from spontaneous Eker rat leiomyomas, we examined the estrogen‐responsive phenotype of these tumor cells. Leiomyoma‐derived ELT cell lines proliferated in response to estrogen, and estrogen‐induced cell proliferation could be inhibited by the estrogen antagonist ICI 182780 and the selective estrogen‐receptor modulators (SERMs) raloxifene and tamoxifen. In addition to inhibiting cell growth, these antagonists also inhibited estrogen‐induced increases in progesterone‐receptor expression. These data indicate that SERMs such as raloxifene and tamoxifen act as estrogen antagonists in uterine myometrial cells and suggest that this class of compounds may be effective for treatment of this important gynecologic neoplasm.

Intermittent administration of parathyroid hormone (1-34) stimulates matrix metalloproteinase-9 (MMP-9) expression in rat long bone.

Intermittent doses of parathyroid hormone (PTH) stimulate bone formation in animals and humans, but the molecular mechanisms underlying this phenomenon are not understood. Bone formation culminates with the expression of type I collagen, osteocalcin, and alkaline phosphatase, but genes that initiate and support the anabolic response are not known. To identify novel PTH‐regulated genes in bone during the anabolic response, we used differential display‐polymerase chain reaction (DDRT‐PCR) to analyze RNA from young male rats injected with either human PTH (1‐34) or vehicle control, once daily for 5 days. Total RNA was isolated from the distal femur metaphysis at 1, 6, and 48 h after the final injection and subjected to DDRT‐PCR. We identified three PTH‐responsive transcripts as matrix metalloproteinase‐9 (MMP‐9), creatine kinase, and the α1(I) polypeptide chain (COL1A1) of type I collagen. The concomitant upregulation of MMP‐9 and COL1A1 during bone formation was particularly intriguing. Further characterization of MMP‐9 expression revealed that it was localized to osteoblasts, osteocytes, megakaryocytes, and cells of the bone marrow in the rat distal femur metaphysis. Northern analysis for MMP‐9 expression in other tissues indicated that this transcript was present in the kidney and brain. In vitro, PTH regulated the protein synthesis of MMP‐9 by osteoblasts of the primary spongiosa. We propose that PTH may promote bone formation by mediating the subtle variation in MMP activities, thus preparing the extracellular matrix for the subsequent bone cell migration and deposition of new osteoid. J. Cell. Biochem. 70:391‐401, 1998.

Analysis of early changes in the articular cartilage transcriptisome in the rat meniscal tear model of osteoarthritis: pathway comparisons with the rat anterior cruciate transection model and with human osteoarthritic cartilage

OBJECTIVE The purpose of this study was to use microarray technology to: (1) understand the early molecular events underlying the damage of articular cartilage initiated by this surgical procedure, and (2) determine whether these changes mimic those that are occurring in human osteoarthritic (OA) cartilage. DESIGN Cartilage was harvested from both medial and lateral sides of the tibial plateaus and femoral condyles of both meniscal tear (MT) and sham surgery groups on days 3, 7 and 21 post-surgery. mRNA prepared from these rat cartilage samples was used for microarray analysis. RESULTS Statistical analysis identified 475 genes that were differentially expressed between the sham and MT groups, at one or more of the time points that were analyzed. By integrating these genes with OA-related genes reported previously in a rat OA model and in human OA array studies, we identified 20 commonly changed genes. Six out of these 20 genes (Col5A1, Col6A2, INHBA, LTBP2, NBL1 and SERPINA1) were differentially expressed in two animal models and in human OA. Pathway analysis identified some key features of OA pathology, namely cartilage extracellular matrix remodeling, angiogenesis, and chondrocyte cell death that were recapitulated in the animal models. The rat models suggested increased inflammation and cholesterol metabolic pathways may play important role in early cartilage degeneration. CONCLUSION We identified a large number of differentially expressed genes in the articular cartilage of the MT model. While there was lack of overall identity in cartilage gene expression between the rat models and human OA, several key biological processes were recapitulated in the rat MT OA model.

Analysis of differential gene expression in rat tibia after an osteogenic stimulus in vivo: Mechanical loading regulates osteopontin and myeloperoxidase

The skeleton has the ability to alter its mass, geometry, and strength in response to mechanical stress. In order to elucidate the molecular mechanisms underlying this phenomenon, differential display reverse transcriptase‐polymerase chain reaction (DDRT‐PCR) was used to analyze gene expression in endocortical bone of mature female rats. Female Sprague‐Dawley rats, approximately 8 months old, received either a sham or bending load using a four‐point loading apparatus on the right tibia. RNA was collected at 1 h and 24 h after load was applied, reverse‐transcribed into cDNA, and used in DDRT‐PCR. Parallel display of samples from sham and loaded bones on a sequencing gel showed several regulated bands. Further analysis of seven of these bands allowed us to isolate two genes that are regulated in response to a loading stimulus. Nucleotide analysis showed that one of the differentially expressed bands shares 99% sequence identity with rat osteopontin (OPN), a noncollagenous bone matrix protein. Northern blot analysis confirms that OPN mRNA expression is increased by nearly 4‐fold, at 6 h and 24 h after loading. The second band shares 90% homology with mouse myeloperoxidase (MPO), a bactericidal enzyme found primarily in neutrophils and monocytes. Semiquantitative PCR confirms that MPO expression is decreased 4‐ to 10‐fold, at 1 h and 24 h after loading. Tissue distribution analysis confirmed MPO expression in bone but not in other tissues examined. In vitro analysis showed that MPO expression was not detectable in total RNA from UMR 106 osteoblastic cells or in confluent primary cultures of osteoblasts derived from either rat primary spongiosa or diaphyseal marrow. Database analysis suggests that MPO is expressed by osteocytes. These findings reinforce the association of OPN expression to bone turnover and describes for the first time, decreased expression of MPO during load‐induced bone formation. These results suggest a role for both OPN and MPO expression in bone cell function. J. Cell. Biochem. 68:355–365, 1998.

Analysis of regulator of G‐protein signaling‐2 (RGS‐2) expression and function in osteoblastic cells

Regulator of G‐protein signaling‐2 (RGS‐2) belongs to a novel family of GTPase‐activating proteins that rapidly turn‐off G‐protein coupled receptor signaling. RGS proteins contain a characteristic RGS domain by which they interact with the α‐subunit of G‐proteins and drive them into their inactive GDP‐bound forms. Previously, we have reported that RGS‐2 mRNA is rapidly and transiently increased by PTH in rat bone and in osteoblast cultures in vitro. In this study, we further explored the molecular basis for the regulation of RGS‐2 by cloning and functionally characterizing the RGS‐2 gene promoter. We cloned 2.3‐ and 2.8‐kb fragments of the 5′‐flanking regions of the rat and mouse RGS‐2 genes, respectively, and generated a stable clone of UMR106 osteoblastic cells containing the rat RGS‐2 promoter driving the β‐gal reporter gene (p2.3RGS‐2‐β‐gal). Treatment of the stable clone with PTH resulted in a maximal 2.2‐ to 3.6‐fold increase in promoter activity at 8 h, reminiscent of the early response observed with endogenous RGS‐2 mRNA regulation. Further, PTH (1–38), (1–31), PTHrP (1–34), and forskolin, which elevate cAMP levels, stimulated the promoter, while PTH (3–34) and (7–34), which do not readily stimulate cAMP accumulation, and PMA that directly activates protein kinase C, had no effect on promoter activity. Taken together, these results implicate the involvement of the Gαs‐adenylate cyclase‐protein kinase A pathway in stimulating RGS‐2 expression. Maintenance of a hyperphosphorylated state via the inhibition of type 2A protein phosphatases by okadaic acid, resulted in a strong dose‐dependent increase in transcriptional activity of the RGS‐2 promoter as well as that of the endogenous RGS‐2 gene. Furthermore, overexpression of the osteoblast‐specific transcription factor Runx2 also led to a stimulation of RGS‐2 promoter activity. Functional analysis using RGS‐2 overexpression suggests the potential negative regulatory effects of RGS‐2 on PTH‐ and forskolin‐induced cAMP production in osteoblastic cells. In summary, our data suggest that PTH treatment results in a direct transcriptional stimulation of RGS‐2 that in turn may play a role in modulating the duration/intensity of PTH receptor signaling. J. Cell. Biochem. 85: 837–850, 2002.

Identification of signal transduction pathways and promoter sequences that mediate parathyroid hormone 1–38 inhibition of osteoprotegerin gene expression

Osteoprotegerin (OPG), a secreted member of the tumor necrosis receptor superfamily, is a potent inhibitor of osteoclast formation and bone resorption. Parathyroid hormone (PTH), a potent inducer of osteoclast formation, suppresses OPG mRNA expression in vitro and in vivo. To determine the molecular basis of this inhibition, we analyzed the effects of PTH on the human OPG promoter (−5917 to +19) fused with β‐galactosidase reporter gene in stable and transient transfections into rat osteoblast‐like UMR106 cells. The effect of PTH on OPG promoter expression was biphasic and concentration‐dependent. PTH (1–100 nM) induced the transcriptional activity of the OPG promoter (1.7‐fold) at 8 h followed by a gradual decrease with maximal inhibition (6.6‐fold) at 24–48 h. To ascertain the signal transduction pathways mediating PTH (1–38) effects on OPG gene expression, we compared the effects of PTH with PTH analogs, parathyroid hormone‐related protein 1–34 (PTHrP 1–34), forskolin, 3‐isobutyl‐1‐methylxanthine (IBMX), dibutyryl cAMP, phorbol‐12‐myristate‐13‐acetate (PMA), thapsigargin and calcium ionophore A23187. PTH 1–31 and PTHrP 1–34, which stimulate the cAMP/PKA pathway, and other activators of cAMP/PKA, forskolin, IBMX, N6, O2′‐dibityryl adenosine 3′,5′‐cyclic monophosphate (dibutyryl cAMP), all elicited a similar biphasic response on OPG promoter expression. PTH analogs PTH 3–34 and PTH 7–34, that do not stimulate cAMP production, had no effect on OPG expression. In contrast, phorbol‐12‐myristate‐13‐acetate (PMA), an activator of PKC, stimulated OPG promoter expression, while thapsigargin and calcium ionophore A23187, which increase intracellular Ca2+, showed a dose‐dependent inhibition of OPG promoter expression. To delineate the promoter sequences that mediate the inhibitory effects of PTH on OPG transcription, we analyzed systematic deletions of the OPG promoter for responsiveness in transient transfection assays. The major inhibitory effects of PTH were localized to 391 bp (−372 to +19) of the proximal promoter. Deletions of the promoter region led to a complete loss of responsiveness. Taken together, these results demonstrate that the inhibitory effects of PTH on OPG are mediated at the transcriptional level through cis elements in the proximal promoter. The similar biphasic response of OPG to PTH, PTH 1–31, PTHrP 1–34, forskolin, IBMX and dibutyryl cAMP suggests that PTH regulates OPG transcription via activation of the cAMP/PKA signal transduction pathway. J. Cell. Biochem. 84: 1–11, 2002.