DACH1 negatively regulates the human RANK ligand gene expression in stromal/preosteoblast cells

Tumor necrosis factor (TNF) family member, the receptor activator of NF-κB ligand (RANKL) is expressed on marrow stromal/osteoblast cells in response to several osteotropic factors and is critical for osteoclast precursor differentiation to form multinucleated osteoclasts, which resorb bone [Lacey et al., 1998; Hsu et al., 1999; Kong et al., 1999]. Enhanced levels of RANKL are associated with pathologic conditions such as Paget's disease of bone [Menaa et al., 2000; Neale et al., 2000] and multiple myeloma [Roux et al., 2002]. Several osteotropic factors such as l,25-(OH)2D3, parathyroid hormone (PTH), interleukin 1β (IL-1β), interleukin-11, and prostaglandin E2 (PGE2) induce osteoclast differentiation through enhanced expression of RANKL in marrow stromal/osteoblast cells [Nakashima et al., 2000; Lee et al., 2002], but the molecular mechanisms which regulate RANKL gene expression are unclear. It has been reported that IL-1β and TNF-α stimulate RANKL expression in human bone marrow stromal cells through activation of p38 MAP kinase pathway [Rossa et al., 2006]. In addition, fibroblast growth factor-2 (FGF-2) has been shown to induce RANKL production through COX-2-mediated prostaglandin synthesis and by suppressing osteoclastogenesis inhibitory factor in mouse osteoblastic cells [Nakagawa et al., 1999]. Similarly, lipopolysaccharide treatment increased the levels of RANKL expression through activation of Toll-like receptors in primary murine osteoblast cells [Kikuchi et al., 2001]. Furthermore, transforming growth factor β (TGF-β) has been shown to increase RANKL expression in activated T-cells by increasing anti-CD3 [Wang et al., 2002]. Recently, it has also been reported that PTH stimulates RANKL expression through cAMP/protein kinase A/CREB cascade [Fu et al., 2006]. We recently demonstrated that heat shock factor-2 (HSF-2) is a downstream target of FGF-2 signaling to induce RANKL expression in bone marrow stromal/preosteoblast cells [Roccisana et al., 2004]. Heat shock proteins (HSP) are molecular chaperones expressed in cells in response to a variety of stimuli such as temperature and stimulation of membrane-bound receptors by hormones/cytokines and other chemical factors. Thus, HSP are an integral part of mammalian development [Christians et al., 2003]. Heat shock transcription factors (HSF), which binds to the heat shock responsive element (HSE), modulate expression of HSP and several other genes including TNF-α family [Mathew et al., 1998; Snoeckx et al., 2001]. Although multiple osteotropic factors including FGF-2 are known to modulate RANKL gene expression in the bone microenvironment, the transcriptional regulatory mechanisms operative in marrow stromal/preosteoblast cells are not well established. DACH1 is a human homolog of the Drosophila dachshund gene, which is a key regulator for organ development and is considered a cell fate determination factor. DACH1 contains a conserved domain (dachshund domain (DS)) in the N-terminal region that is structurally homologous with the Ski and Sno proto-oncogenes and interacts with the nuclear co-repressor NCoR to modulate transcription factor activity. We have previously reported that DACH1 represses TGF-β signaling through binding to Smad4 [Wu et al., 2003]. Two vertebrate homologues, Dach1 and Dach2 are partially functionally redundant, since Dach1−/− mice survive to birth but exhibit postnatal lethality associated with a failure to suckle, cyanosis, and respiratory distress [Davis et al., 2001]. DACH1 expression is lost in a subset of human breast cancers associated with poor prognosis. Reintroduction of DACH1 inhibits breast tumor cell proliferation and tumor growth through suppression of cyclin D1 [Wu et al., 2006]. DACH1 inhibits TGF-β signaling in ovarian cancer cells [Sunde et al., 2006]. These studies are part of a growing body of evidence that DACH1 may function as a tumor suppressor. Recent evidence indicates that DACH1 is associated with FGF signaling during limb and skeletal development and may regulate cell proliferation or differentiation [Horner et al., 2002]. Dach1 binds with Runx2/Cbfa1 and co-localizes with cyclin-dependent kinase inhibitor p27 (Kip1) and p57 (Kip2) at the growth plate region in chondrocytes [Horner et al., 2002]. In this study, we show that DACH1 negatively regulate RANKL gene expression in human bone marrow-derived stromal/preosteoblast cells and that FGF-2 signaling promotes HSF-2 binding to DACH1 to modulate RANKL gene expression in these cells.