Immune cells are thought to play an important role in bone loss caused by inflammation (1, 2). Disabling joint destruction in rheumatoid arthritis and the loss of teeth in periodontal disease are examples of skeletal loss that can occur with inflammation. Resorption of bone by osteoclasts represents the primary mechanism responsible for the loss of bone caused by inflammatory disease.Key factors regulating osteoclastogenesis include macrophage colony-stimulating factor (M-CSF) 2 and receptor activator of nuclear factor-B ligand (RANKL). M-CSF is a secreted product of stromal cells/ osteoblasts that enhances colony expansion of monocyte/osteoclast progenitor cells. RANKL exists both as a transmembrane protein in osteoblasts/stromal cells and as a soluble protein (3-6). RANKL directs the expanded progenitor cell population to the osteoclast lineage by activation of receptor activator of nuclear factor-B (RANK). The interaction between RANKL and RANK can be inhibited by osteoprotegerin (OPG), a decoy receptor released from stromal cells/osteoblasts.RANKL stimulation of RANK causes receptor trimerization and recruitment of tumor necrosis factor receptor-associated factors (TRAFs). TRAF1, 2, 3, and 5 bind to the carboxyl-terminal end of the RANK trimer, whereas TRAF6 binds more closely to the membrane. Downstream intracellular signaling mediated by RANK in osteoclast progenitor cells includes TRAF6-dependent activation of NF-B, mitogen-activated protein kinases (MAP kinases) and AP-1, and activation of c-Src and the phosphatidylinositol 3-kinase/Akt pathway (5-7). In addition, immunoreceptor tyrosine-based activation motif-mediated costimulatory signals have been shown to be required for expression of nuclear factor of activated T-cells 2 (NFAT2), the transcription factor believed to be crucial for osteoclast differentiation (8, 9). Several of these intracellular signaling molecules, including p50/p65, c-Fos, NFAT2, Fc receptor common ␥ subunit (FcR␥)/DNAX-activation protein 12 kD (DAP 12), and TRAF6, as well as RANK, RANKL, OPG, M-CSF, and the M-CSF receptor c-Fms, have been shown by gene deletion studies to be essential for osteoclastogenesis (2, 7, 10 -14).* This work was supported by grants from the Swedish Science Council (project 07525), the Swedish Rheumatism Association, the Royal 80 Year Fund of King Gustav V, the Swedish Dental Society, and the County Council of Västerbotten. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom correspondence should be addressed. Tel.: 46-90-785-6290; Fax: 46-90-139289; E-mail: ulf.lerner@odont.umu.se.2 The abbreviations used are: M-CSF, macrophage colony-stimulating factor; ALP, alkaline phosphatase; ␣-MEM, ␣-modification of minimum essential medium; BMM, bone marrow macrophages; CT, calcitonin; CTR, calcitonin receptor; DAP12, DNAX-activating protein 12; D3, 1,25(OH) 2 -vitamin D 3 ; ERK, e...

Whether vitamin A promotes skeletal fragility, has no effect on fracture rate, or protects against bone loss is unclear. In the present study, effects of retinoids on osteoclast differentiation in cultured mouse bone marrow cells (BMCs), bone marrow macrophages (BMMs), spleen cells, and RAW264.7 cells were evaluated by analyzing osteoclast formation and expression of genes important in signal transduction and osteoclast function. All-trans-retinoic acid (ATRA) did not stimulate osteoclastogenesis in BMCs, but inhibited hormone and RANKL-induced gene expression and formation of osteoclasts. In BMMs, spleen cells, and RAW264.7 cells, osteoclast differentiation and formation stimulated by M-CSF/RANKL were inhibited (IC(50) = 0.3 nM) by ATRA. The effect was exerted at an early step of RANKL-induced differentiation. ATRA also abolished increases of the transcription factors c-Fos and NFAT2 stimulated by RANKL and suppressed down-regulation of the antiosteoclastogenic transcription factor MafB. By comparing effects of several compounds structurally related to ATRA, as well as by using receptor antagonists, evaluation pointed to inhibition being mediated by RARalpha, with no involvement of PPARbeta/delta. The results suggest that activation of RARalpha by retinoids in myeloid hematopoietic precursor cells decreases osteoclast formation by altering expression of the transcription factors c-Fos, NFAT2, and MafB.

No abstract

The recently cloned PMP22 gene, the rat variant of the murine growth arrest-specific gene gas3, encodes a new 22 kD integral membrane glycoprotein of peripheral myelin. By means of in situ hybridization and immunohistochemistry, we have (1) analyzed PMP22 expression in myelinated and nonmyelinated peripheral nerves, and (2) compared the spatio-temporal changes in the expression of PMP22 mRNA with the expression of the myelin genes P0 and MBP (myelin basic protein) in developing as well as degenerating and regenerating sciatic nerve of rat. (3) We further investigated the expression of PMP22 mRNA by Northern blot in cultured Schwann cells maintained under different conditions of cell growth and arrest. Expression of PMP22 mRNA is restricted to Schwann cells of myelinated peripheral nerve. Transection of sciatic nerve in adult rat leads to a simultaneous and rapid decline in both PMP22 and P0 mRNA to nondetectable levels in the degenerating distal stump. When a demyelinated and axon-free distal stump, as indicated by the lack of MBP and neurofilament immunoreactivity, was reanastomosed to its proximal counterpart, the coordinated reexpression of PMP22 and MBP succeeded axonal regeneration through the distal segment with a delay of 1-2 weeks. As in regenerating nerve, a striking synchrony of expression of PMP22 and P0 transcripts, as well as MBP immunoreactivity, could be observed during sciatic nerve development. Further, in vitro evidence suggests that, unlike NIH3T3-fibroblasts, expression of PMP22/gas3 is not strictly growth arrest-specific in Schwann cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Receptor activator of NF-kappaB ligand (RANKL) is crucial in osteoclastogenesis but signaling events involved in osteoclast differentiation are far from complete and other signals may play a role in osteoclastogenesis. A more direct pathway for cellular crosstalk is provided by gap junction intercellular channel, which allows adjacent cells to exchange second messengers, ions, and cellular metabolites. Here we have investigated the role of gap junction communication in osteoclastogenesis in mouse bone marrow cultures. Immunoreactive sites for the gap junction protein connexin 43 (Cx43) were detected in the marrow stromal cells and in mature osteoclasts. Carbenoxolone (CBX) functionally blocked gap junction communication as demonstrated by a scrape loading Lucifer Yellow dye transfer technique. CBX caused a dose-dependent inhibition (significant > or = 90 microM) of the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells formed in 7- to 8-day marrow cultures stimulated by parathyroid hormone (PTH; 10 nM) or forskolin (FSK; 1 microM). Furthermore, CBX (100 microM) significantly inhibited prostaglandin E2 (PGE2; 10 microM) and 1,25(OH)2-vitamin D3 stimulated osteoclast differentiation in the mouse bone marrow cultures. Consequently, quantitative real-time polymerase chain reaction (PCR) analysis demonstrated that CBX downregulated the expression of osteoclast phenotypic markers, but without having any significant effects on RANK, RANKL, and osteoprotegerin (OPG) mRNA expression. However, the results demonstrated that CBX significantly inhibits RANKL-stimulated (100 ng/ml) osteoclastogenesis in the mouse bone marrow cultures. Taken together, our results suggests that gap junctional diffusion of messenger molecules interacts with signaling pathways downstream RANKL in osteoclast differentiation. Further studies are required to define the precise mechanisms and molecular targets involved.