Faculty Bibliography

Intermittent parathyroid hormone (PTH) administration increases bone formation and bone mass and is being used as a therapy for osteoporosis. By contrast, chronic hyperparathyroidism results in the metabolic bone disease osteitis fibrosa characterized by local bone resorption and peritrabecular bone marrow fibrosis in humans. The animal models that can mimic the paradoxical effects of PTH provide the basis for further study of the functions of this hormone in skeletal tissues. In both rats and mice, the anabolic effects of PTH on bone can be achieved by daily injections subcutaneously and the catabolic effects can be achieved by continuous infusion with osmotic pumps. This chapter offers detailed information, such as the dosage and preparation of PTH, using the example of treatment of rats with PTH intermittently or continuously. High-quality, RT-PCR ready RNA is required for the analysis of gene expression. For the analysis of gene expression in bone, usually long bones are used for RNA extraction. Here we describe how to extract RNAs from the metaphyseal trabecular primary spongiosa of rat femur by a method based on two commercially available kits. This protocol can be used in other tissues with slight modification of the amount of reagent used according to the tissue size.

Parathyroid hormone (PTH) stimulates bone formation when injected daily but causes severe bone loss with continuous infusion. The mechanism of its paradoxical effects is still elusive. In this study, we compared changes in the gene expression profile in bone induced by intermittent or continuous treatment with three different PTH peptides, PTH-(1-34), -(1-31), and -(3-34), in Sprague-Dawley female rats. PTH-(1-34) regulated numerous genes (approximately 1,000), but differentially, in both regimes. PTH-(1-31) regulated a similar number of genes in the intermittent regimen but fewer in the continuous regimen, consistent with its less potent catabolic effect. PTH-(3-34) regulated very few genes in both regimes, which suggests the protein kinase C pathway plays a limited role in mediating the dual effects of PTH, whereas the cAMP-dependent protein kinase A pathway appears to predominate. In the intermittent treatment, many genes encoding signaling mediators, transcription factors, cytokines, and proteases/protease inhibitors are regulated rapidly and cyclically with each PTH injection; genes associated with skeletal development show a slowly accruing pattern of expression. With continuous treatment, some genes are regulated from 6 h, and the mRNA levels are sustained with a longer infusion, whereas others show a kinetic decrease and then increase later. Significant up-regulation of genes stimulating osteoclastogenesis in the anabolic regime suggests a provocative and paradoxical theme for the anabolic effect of PTH that a full anabolic response requires a transient up-regulation of genes classically associated with a resorptive response. Ingenuity pathway analysis was performed on the microarray data. A novel signaling network was established that is differentially regulated in the two PTH treatment regimes. Key regulators are suggested to be AREG, CCL2, WNT4, and cAMP-responsive element modulator

The clinical findings that alendronate blunted the anabolic effect of human parathyroid hormone (PTH) on bone formation suggest that active resorption is involved and enhances the anabolic effect. PTH signals via its receptor on the osteoblast membrane, and osteoclasts are impacted indirectly via the products of osteoblasts. Microarray with RNA from rats injected with human PTH or vehicle showed a strong association between the stimulation of monocyte chemoattractant protein-1 (MCP-1) and the anabolic effects of PTH. PTH rapidly and dramatically stimulated MCP-1 mRNA in the femora of rats receiving daily injections of PTH or in primary osteoblastic and UMR 106-01 cells. The stimulation of MCP-1 mRNA was dose-dependent and a primary response to PTH signaling via the cAMP-dependent protein kinase pathway in vitro. Studies with the mouse monocyte cell line RAW 264.7 and mouse bone marrow proved that osteoblastic MCP-1 can potently recruit osteoclast monocyte precursors and facilitate receptor activator of NF-kappaB ligand-induced osteoclastogenesis and, in particular, enhanced fusion. Our model suggests that PTH-induced osteoblastic expression of MCP-1 is involved in recruitment and differentiation at the stage of multinucleation of osteoclast precursors. This information provides a rationale for increased osteoclast activity in the anabolic effects of PTH in addition to receptor activator of NF-kappaB ligand stimulation to initiate greater bone remodeling

Parathyroid hormone (PTH) functions as an essential regulator of calcium homeostasis and as a mediator of bone remodeling. We have already shown that PTH stimulates the expression of matrix metalloproteinase-13 (MMP-13), which is responsible for degrading components of extracellular matrix. We have hypothesized that histone deacetylases (HDACs) are involved with PTH-induced MMP-13 gene expression in the osteoblastic cell line, UMR 106-01. We have shown that PTH profoundly regulates HDAC4 in UMR 106-01 cells through a PKA-dependent pathway, leading to removal of HDAC4 from the MMP-13 promoter and its enhanced transcription. Understanding the mechanism of how HDACs affect osteoblast differentiation and mineralization will identify new theraupeutic methods for bone diseases, such as osteoporosis and multiple myeloma

In bone, parathyroid hormone (PTH) exerts either a catabolic or an anabolic effect depending on its method of administration. This paradoxical action has led to the use of PTH as an effective treatment for osteoporosis. The Wnt family of signaling proteins has a critical role in multiple events, which are necessary for proper animal development and survival, yet their exact method of action in bone remains elusive. We have uncovered a novel link between Wnt-4 and PTH. We think that, in bone, Wnt-4 signaling in response to PTH implicates cross-talk of multiple signaling pathways. This work hopes to further elucidate Wnt signaling in bone and provide greater understanding of PTH's anabolic effects in bone

Epidermal growth factor (EGF)-like ligands and their receptors constitute one of the most important signaling networks functioning in normal tissue development and cancer biology. Recent in vivo mouse models suggest this signaling network plays an essential role in bone metabolism. Using a coculture system containing bone marrow macrophage and osteoblastic cells, here we report that EGF-like ligands stimulate osteoclastogenesis by acting on osteoblastic cells. This stimulation is not a direct effect because osteoclasts do not express functional EGF receptors (EGFRs). Further studies reveal that EGF-like ligands strongly regulate the expression of two secreted osteoclast regulatory factors in osteoblasts by decreasing osteoprotegerin (OPG) expression and increasing monocyte chemoattractant protein 1 (MCP1) expression in an EGFR-dependent manner and consequently stimulate TRAP-positive osteoclast formation. Addition of exogenous OPG completely inhibited osteoclast formation stimulated by EGF-like ligands, while addition of a neutralizing antibody against MCP-1 exhibited partial inhibition. Coculture with bone metastatic breast cancer MDA-MB-231 cells had similar effects on the expression of OPG and MCP1 in the osteoblastic cells, and those effects could be partially abolished by the EGFR inhibitor PD153035. Because a high percentage of human carcinomas express EGF-like ligands, our findings suggest a novel mechanism for osteolytic lesions caused by cancer cells metastasizing to bone

Bone morphogenetic proteins (BMPs) strongly promote osteoblast differentiation. Pulsed electromagnetic fields (PEMFs) promote fracture healing in non-union fractures. In this study, we hypothesized that a combined BMP-2 and PEMF stimulation would augment bone formation to a greater degree than treatment with either single stimulus. BMP-2 maximally increased the proliferative activity of rat primary osteoblastic cells at 25 ng/ml concentration. Real-time reverse transcription-polymerase chain reaction (RT-PCR) showed that BMP-2 stimulated mRNA levels of alkaline phosphatase (ALP), alpha(1) (I) procollagen, and osteocalcin (OC) in the differentiation phase and only OC mRNA expression in the mineralization phase after 24-h treatment. Both BMP-2 and PEMF (Spinal-Stim) increased cell proliferation, which was additive when both agents were combined. PEMF alone or together with BMP-2 increased only ALP mRNA expression and only during the differentiation phase 24 h after one 4-h treatment. This effect was additive when both agents were combined. Continuous daily 4-h treatment with PEMF alone or together with BMP-2 increased expression of all three osteoblast marker genes during the differentiation phase and increased the mineralized matrix. This effect was additive when both agents were combined, suggesting that the two interventions may be working on different cellular pathways. Thus, a combined effect of BMP-2 and PEMF in vitro could be considered as groundwork for in vivo bone development that may support skeletal therapy

We report on the role of K+ currents in the mechanisms regulating the proliferation of UMR 106-01 osteoblastic osteosarcoma cells. Electrophysiological analysis showed that UMR 106-01 cells produce robust K+ currents that can be pharmacologically separated into two major components: a E-4031-susceptible current, I E-4031, and a tetraethylammonium (TEA)-susceptible component, I TEA. Western blot and RT-PCR analysis showed that I E-4031 is produced by ether a go-go (eag)-related channels (ERG). Incubation of the cells with E-4031 enhanced their proliferation by 80%. Application of E-4031 in the bath solution did not induce instantaneous changes in the membrane resting potential or in the level of cytosolic calcium; however, the cells were slightly depolarized and the calcium content was significantly increased upon prolonged incubation with the compound. Taken together these findings indicate that ERG channels can impair cell proliferation. This is a novel finding that underscores new modes of regulation of mitosis by voltage-gated K+ channels and provides an unexpected insight into the current view of the mechanisms governing bone tissue proliferation