Nmp4/CIZ suppresses parathyroid hormone-induced increases in trabecular bone.

An accumulating body of clinical studies demonstrates that once-daily intermittent doses of human parathyroid hormone (1–34) [hPTH (1–34)] reduce the fracture risk in patients with severe osteoporosis (Dempster et al., 2001; Neer et al., 2001). Despite its proven efficacy in reducing fractures, and subsequent FDA approval as an osteoporosis therapy, the cellular and molecular mechanisms of action employed by intermittent PTH remain to be fully elucidated. Upon binding to its receptor (PTHR1), PTH activates a multitude of osteoblast molecular signaling networks, beginning with the cAMP/PKA and the cytosolic calcium/PKC response limbs. Subsequent downstream targets include the activation of the transcription factors CREB and Runx2, downregulation of the osteocyte-derived bone formation inhibitor sclerostin, and initiation of an IGF-1 autocrine/paracrine signaling loop, among others (Feister et al., 2000; Bikle et al., 2002; Swarthout et al., 2002; Hurley et al., 2006; Yakar et al., 2006; Zhang et al., 2006; Leupin et al., 2007; Li et al., 2007; Merciris et al., 2007). The collective end result of these and other PTH-induced networks is the enhanced differentiation of committed osteoblast precursors in the bone marrow, and prolongation of the lifespan of the mature osteoblast via suppressed apoptosis (reviewed in Jilka, 2007; Martin and Seeman, 2007). While much of the investigation of anabolic PTH signal transduction and molecular regulation has been focused on the activation of osteogenic kinases, secretion of osteogenic ligands and ECMs, and expression of osteogenic genes, little is known about the inhibitory mechanisms on bone formation that are also activated by the anabolic signaling events. These mechanisms, though antagonistic to the bone-building effects of the signal, likely serve as an important negative feedback mechanism to prevent hyper-responsiveness to the signal. For example, the anti-apoptotic effect of PTH on osteoblasts requires Runx2-driven transcription of survival genes but PTH also induces the Smurf1-mediated proteasomal degradation of this trans-acting protein (Bellido et al., 2003). This mechanism appears to support a self-limiting response of the hormone’s own anabolic action, though it has not been tested in vivo. Similarly, up-regulation of the CREM gene, the products of which act as transcriptional attenuators, may keep the anabolic response to intermittent PTH in check, by restraining PTH-induced osteoclastogenesis (Liu et al., 2007). Elucidation of the osteo-inhibitory mechanisms that are activated by anabolic PTH could yield an attractive target for osteoporosis therapy, either alone or in conjunction with PTH treatment, by essentially providing a means to suppress the “off signal” that accompanies each anabolic activation cycle. We have potentially identified such a target in a series of experiments conducted over the last 12 years (Alvarez et al., 1997, 1998, 2005; Thunyakitpisal et al., 2001; Shah et al., 2004). Nuclear matrix protein 4/cas interacting zinc finger protein (Nmp4/CIZ) is a trans-acting nucleocytoplasmic shuttling protein that, in osteoblasts, acts as a transcriptional attenuator (Shen et al., 2002; Shah et al., 2004; Morinobu et al., 2005). Nmp4/CIZ suppresses PTH-driven matrix metalloproteinase-13 (MMP-13) gene transcription in MC3T3-E1 osteoblast-like cells (Shah et al., 2004). Similarly BMP2-induced up-regulation of alkaline phosphatase, osteocalcin, and type I collagen are all suppressed in MC3T3-E1 cells over-expressing Nmp4/CIZ (Shen et al., 2002), and BMP2- induced bone formation on adult calvariae in vivo was enhanced by CIZ deficiency (Morinobu et al., 2005). Collectively, our studies indicate that Nmp4/CIZ activation induced by anabolic regimens of PTH might be a mechanism to partially antagonize the osteogenic response of bone tissue to therapeutic doses of PTH. From these data, an obvious question emerges: does the anabolic response to PTH improve when Nmp4/CIZ is disabled/deleted? If so, Nmp4/CIZ might represent an attractive pharmacologic target for inhibition in the search for new osteoporosis therapies. In the present communication, we examine the role of Nmp4/CIZ in normal skeletal homeostasis and in regulating the response of the skeleton to intermittent PTH. We engineered mice with a loss-of-function mutation in Nmp4/CIZ, and measured bone mass, density, structure, and gene expression, at several time points. We further evaluated the ramifications of the null mutation on anabolic PTH signaling by challenging the mice with intermittent PTH for 7 weeks. Nmp4-null mice exhibited a modest gain in skeletal BMD and BMC between 8 and 16 weeks of age as compared to WT animals. Notably, the Nmp4-knockout (KO) animals displayed a significantly enhanced hormone-induced gain in femoral cancellous bone. The present data and our previous in vitro studies support our hypothesis that Nmp4/CIZ is a key component to an inhibitory mechanism of bone formation. As such, it appears to act as a transcriptional attenuator that suppresses osteoid synthesis thus limiting anabolic PTH-mediated gains in cancellous bone.