Determination of Dual Effects of Parathyroid Hormone on Skeletal Gene Expression in Vivo by Microarray and Network Analysis

Abstract

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 (∼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. 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 (∼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. PTH, 2The abbreviations used are: PTH, parathyroid hormone; h, human; RT, reverse transcription; ELISA, enzyme-linked immunosorbent assay; RANKL, receptor activator of NF-κB ligand; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; OPG, osteoprotegrin; IPA, Ingenuity pathway analysis; FSH, follicle-stimulating hormone. 2The abbreviations used are: PTH, parathyroid hormone; h, human; RT, reverse transcription; ELISA, enzyme-linked immunosorbent assay; RANKL, receptor activator of NF-κB ligand; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; OPG, osteoprotegrin; IPA, Ingenuity pathway analysis; FSH, follicle-stimulating hormone. administered intermittently, stimulates bone formation in contrast to antiresorptive agents, which reduce bone resorption. Concerns with the limited improvement of antiresorptive treatments in severe osteoporosis led to a search for alternative anabolic agents (1Reeve J. Meunier P.J. Parsons J.A. Bernat M. Bijvoet O.L. Courpron P. Edouard C. Klenerman L. Neer R.M. Renier J.C. Slovik D. Vismans F.J. Potts Jr., J. Br. Med. J. 1980; 280: 1340-1344Crossref PubMed Scopus (519) Google Scholar). Teraparatide, recombinant human parathyroid hormone consisting of the first 34 of the 84 amino acids of human parathyroid hormone, has been shown to reduce significantly the risk of both vertebral and nonvertebral fractures in postmenopausal women and to significantly increase bone mineral density (2Cappuzzo K.A. Delafuente J.C. Ann. Pharmacother. 2004; 38: 294-302Crossref PubMed Scopus (38) Google Scholar). It has become the only anabolic agent currently approved for the treatment of osteoporosis in the United States. Bone is a highly specialized form of connective tissue and dynamic organ in all higher vertebrates; it undergoes continuous regeneration. Bone remodeling occurs at discrete sites within the skeleton and proceeds in an orderly fashion, with coupling of osteoclastic resorption and osteoblastic formation (3Young M.F. Kerr J.M. Ibaraki K. Heegaard A.M. Robey P.G. Clin. Orthop. Relat. Res. 1992; 281: 275-294PubMed Google Scholar). This physiological process is coordinated and tightly regulated by local and endocrine factors to ensure that there is a balance between bone formation and bone resorption (4Marks S.C. Hermey D.C. Bilezikian J.P. Raisz L.G. Rodan G.A. Principles of Bone Biology. 1996: 3-14Google Scholar). PTH is known to regulate bone metabolism directly by its receptor on the osteoblast plasma membrane; binding of PTH to its receptor causes release of cytokines and growth factors from the osteoblast. As a result, it indirectly affects osteoclasts and other bone cells in a paracrine J.A. PubMed Scopus Google S.C. K. PubMed Scopus Google Scholar). binding to its signaling associated with transient in PTH levels are to the anabolic response. associated with PTH to the catabolic response and bone the of two intermittent continuous treatment, is for the that parathyroid bone bone A of the dynamic of gene expression by PTH is a to the mechanism of its dual anabolic in response to intermittent or continuous treatment of this hormone in been many genes that are regulated by PTH in osteoblastic at the this was there established for the differentially gene from PTH in anabolic catabolic the of the dynamic of gene expression by PTH is to the mechanism of its dual in The of this was to established in PTH anabolic catabolic treatment to genes that bone anabolic catabolic effects of the hormone. the of the and the pathway in mediating the anabolic effects of PTH is and less is known the of the pathway in mediating the effects of on the full PTH PTH-(1-34), or PTH and signaling or and the PTH-(3-34) to it the but a response J. 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Res. PubMed Scopus Google Scholar). a network to in the of known and has been This has been used to changes in gene expression in human an R.M. J.P. C. PubMed Scopus Google and other D. J. J. Clin. Res. PubMed Scopus Google L. J. 280: PubMed Scopus Google Scholar). 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