IntroductionThe osteoclast, the exclusive bone resorptive cell, is derived from hematopoietic stem cells through the common myeloid progenitor to the colony-forming unit for granulocytes and macrophages to the colony-forming unit for macrophages and into the osteoclast lineage. 1 Osteoclast differentiation, function, and survival are regulated by several exogenous cytokines (macrophage colonystimulating factor [M-CSF], receptor activator of nuclear factor B ligand [RANKL], tumor necrosis factor ␣, interleukin-1, and interleukin-6) and hormones (sex steroids, parathyroid hormone, vitamin D, insulinlike growth factor-1, calcitonin, and prostaglandins) through several transcription factors activities identified by studies in genetically engineered mice. 2,3 The transcription factor c-Fos plays an important role in osteoclastogenesis because mice lacking c-Fos develop osteopetrosis. 2-4 c-Fos is not required for normal osteoprogenitor development but is required for osteoclastogenesis. [2][3][4] Nuclear factor of activated T cells cytoplasmic 1 (NFATc1) is also a critical transcription factor that was discovered as an early RANKL-inducible gene by gene expression profiling after RANKL stimulation. 3 NFATc1 molecules act as cofactors with activator protein-1 (AP-1) composed of Fos/Jun proteins to bind to regulatory cis DNA elements, 5 and osteoclastspecific markers such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K have multiple sites recognized by NFATc1 as well as its partner AP-1. 5 MicroRNAs (miRs), which regulate gene expression, are transcribed as primary miRs (pri-miRs) containing a 5Ј-cap structure and poly(A) tail that are processed to produce the mature miRs. 6 Two nuclease enzymes, the nuclear RNaseIII Drosha and the cytosolic RNaseIII Dicer, are known to act sequentially to trim the miRs to mature form. 6 Hundreds of miR genes have been identified in the human genome, and it is estimated that one-third of protein-coding genes are regulated by miRs. Hence, miRs constitute one of the most abundant classes of gene-regulatory molecules in animals, and they are implicated in almost every biologic process, including development timing, cell differentiation, cell proliferation, cell death, metabolic control, transposon silencing, and antiviral defense. 6 Emerging evidence indicates that Dicer-generated miRs play important roles in osteoblastogenesis, chondrocyte proliferation and differentiation, and osteoclastogenesis. [7][8][9][10] Recent studies have shown that Ն 12 miRs, miR-26a, miR-125b, miR-133, miR-135, miR-29a, miR-141, miR-200a, miR-210, miR-29, miR-378, miR-2861, and miR-206, are implicated in osteoblast differentiation. [11][12][13][14][15][16][17][18][19][20] In particular, miR-2861 was identified as a novel miR expressed in mouse osteoblasts promoting osteoblast differentiation by suppressing expression of histone deacetylase 5 at the posttranslational level and contributing to bone formation. 19 Moreover, mutation of miR-2861 causes osteoporosis in humans, suggesting that miR-2861...

Micro-RNAs (miRNAs) are important in regulating cell fate determination because many of their target mRNA transcripts are engaged in cell proliferation, differentiation, and apoptosis. DGCR8, Dicer, and Ago2 are essential factors for miRNA homeostasis. Here we show that these three factors have critical roles in osteoclast differentiation and function. Gene silencing of DGCR8, Dicer, or Ago2 by small interfering RNA revealed global inhibition of osteoclast transcription factor expression and function, decreased osteoclastogenesis, and decreased bone resorption in vitro. In vivo, CD11b؉ -cre/Dicer-null mice had mild osteopetrosis caused by decreased osteoclast number and bone resorption. These results suggest that miRNAs play important roles in differentiation and function of osteoclasts in vitro and in vivo. We found a novel mechanism mediating these results in which PU.1, miRNA-223, NFI-A, and the macrophage colony-stimulating factor receptor (M-CSFR) are closely linked through a positive feedback loop. PU.1 stimulates miRNA-223 expression, and this up-regulation is implicated in stimulating differentiation and function of osteoclasts through negative regulation of NFI-A levels. Down-regulation of NFI-A levels is important for expression of the M-CSFR, which is critical for osteoclast differentiation and function. NFI-A overexpression decreased osteoclast formation and function with down-regulation of M-CSFR levels. Forced expression of the M-CSFR in M-CSF-dependent bone marrow macrophages from Dicer-deficient mice rescued osteoclast differentiation with up-regulation of PU.1 levels. Our studies provide new molecular mechanisms controlling osteoclast differentiation and function by the miRNA system and specifically by miRNA-223, which regulates NFI-A and the M-CSFR levels.

In chronic kidney disease, vascular calcification, renal osteodystrophy, and phosphate contribute substantially to cardiovascular risk and are components of CKD-mineral and bone disorder (CKD-MBD). The cause of this syndrome is unknown. Additionally, no therapy addresses cardiovascular risk in CKD. In its inception, CKD-MBD is characterized by osteodystrophy, vascular calcification, and stimulation of osteocyte secretion. We tested the hypothesis that increased production of circulating factors by diseased kidneys causes the CKD-MBD in diabetic mice subjected to renal injury to induce stage 2 CKD (CKD-2 mice). Compared with non-CKD diabetic controls, CKD-2 mice showed increased renal production of Wnt inhibitor family members and higher levels of circulating Dickkopf-1 (Dkk1), sclerostin, and secreted klotho. Neutralization of Dkk1 in CKD-2 mice by administration of a monoclonal antibody after renal injury stimulated bone formation rates, corrected the osteodystrophy, and prevented CKD-stimulated vascular calcification. Mechanistically, neutralization of Dkk1 suppressed aortic expression of the osteoblastic transcription factor Runx2, increased expression of vascular smooth muscle protein 22-a, and restored aortic expression of klotho. Neutralization of Dkk1 did not affect the elevated plasma levels of osteocytic fibroblast growth factor 23 but decreased the elevated levels of sclerostin. Phosphate binder therapy restored plasma fibroblast growth factor 23 levels but had no effect on vascular calcification or osteodystrophy. The combination of the Dkk1 antibody and phosphate binder therapy completely treated the CKD-MBD. These results show that circulating Wnt inhibitors are involved in the pathogenesis of CKD-MBD and that the combination of Dkk1 neutralization and phosphate binding may have therapeutic potential for this disorder. CKD is a pandemic affecting 26 million Americans in its early stages. 1 CKD is associated with high rates of cardiovascular mortality, making it much more likely that affected individuals will sustain cardiovascular morbidity, including death, than reach end stage kidney disease that requires RRT.

Hyperphosphatemia and vascular calcification have emerged as cardiovascular risk factors among those with chronic kidney disease. This study examined the mechanism by which phosphorous stimulates vascular calcification, as well as how controlling hyperphosphatemia affects established calcification. In primary cultures of vascular smooth muscle cells derived from atherosclerotic human aortas, activation of osteoblastic events, including increased expression of bone morphogenetic protein 2 (BMP-2) and the transcription factor RUNX2, which normally play roles in skeletal morphogenesis, was observed. These changes, however, did not lead to matrix mineralization until the phosphorus concentration of the media was increased; phosphorus stimulated expression of osterix, a second critical osteoblast transcription factor. Knockdown of osterix with small interference RNA (siRNA) or antagonism of BMP-2 with noggin prevented matrix mineralization in vitro. Similarly, vascular BMP-2 and RUNX2 were upregulated in atherosclerotic mice, but significant mineralization occurred only after the induction of renal dysfunction, which led to hyperphosphatemia and increased aortic expression of osterix. Administration of oral phosphate binders or intraperitoneal BMP-7 decreased expression of osterix and aortic mineralization. It is concluded that, in chronic kidney disease, hyperphosphatemia stimulates an osteoblastic transcriptional program in the vasculature, which is mediated by osterix activation in cells of the vascular tunica media and neointima. Chronic kidney disease (CKD) is a fatal illness, and cardiovascular complications are the major causes of morbidity and mortality. 1,2 The causes of the excess cardiovascular mortality associated with CKD are unknown, because the role of the standard risk factors associated with cardiovascular mortality do not account for the increased risk in CKD. 2 There is strong epidemiologic evidence that serum phosphorus is an independent risk factor for cardiovascular events and mortality in CKD. 3,4 The serum phosphorus has been linked to another cardiovascular risk factor, vascular calcification (VC), 3,5,6 an important cause of vascular stiffness in CKD leading to increased pulse wave velocity, increased cardiac work, left ventricular hypertrophy, and decreased coronary artery blood flow. 6 -8 Phosphorus has been further implicated as a cause of VC through studies in vitro that have demonstrated that it induces phenotypic changes in vascular smooth muscle cells (VSMC) by increasing gene transcription of proteins involved in osteoblast function-bone formation 9 and stimulating matrix mineralization. 10 -12 In the uremic calcifying environment, expression of the contractile proteins of VSMC, such as ␣-smooth muscle actin, SM22,

The chronic kidney disease-mineral and bone disorder (CKD-MBD) syndrome is an extremely important complication of kidney diseases. Here we tested whether CKD-MBD causes vascular calcification in early kidney failure by developing a mouse model of early CKD in a background of atherosclerosis stimulated arterial calcification. CKD equivalent in glomerular filtration reduction to human CKD stage 2 stimulated early vascular calcification and inhibited the tissue expression of α-klotho (klotho) in the aorta. In addition, osteoblast transition in the aorta was stimulated by early CKD as shown by the expression of the critical transcription factor, RUNX2. The ligand associated with the klotho-fibroblast growth factor receptor complex, FGF23, was found to be expressed in the vascular media of sham operated mice. Its expression was decreased in early CKD. Increased circulating levels of the osteocyte secreted proteins, FGF23, and sclerostin may have been related to increased circulating klotho levels. Finally, we observed low turnover bone disease with a reduction in bone formation rates more than bone resorption. Thus, the CKD-MBD, characterized by cardiovascular risk factors, vascular calcification, increased circulating klotho, FGF23 and sclerostin levels, and low turnover renal osteodystrophy, was established in early CKD. Early CKD caused a reduction of vascular klotho, stimulated vascular osteoblastic transition, increased osteocytic secreted proteins, and inhibited skeletal modeling producing the CKD-MBD.