Connexin43 in mesenchymal lineage cells regulates body adiposity and energy metabolism in mice.

Connexin43 (Cx43) is the most abundant gap junction protein present in the mesenchymal lineage. In mature adipocytes, Cx43 mediates white adipose tissue (WAT) beiging in response to cold exposure and maintains the mitochondrial integrity of brown adipose tissue (BAT). We found that genetic deletion of Gja1 (Cx43 gene) in cells that give rise to chondro-osteogenic and adipogenic precursors driven by the Dermo1/Twist2 promoter led to lower body adiposity and partial protection against the weight gain and metabolic syndrome induced by a high-fat diet (HFD) in both sexes. These protective effects were related to increased locomotion, fuel utilization, energy expenditure, nonshivering thermogenesis, and better glucose tolerance in conditionally Gja1-ablated mice. Accordingly, Gja1-mutant mice exhibited reduced adipocyte hypertrophy, partially preserved insulin sensitivity, increased BAT lipolysis, and decreased whitening under HFD. This metabolic phenotype was not reproduced with more restricted Gja1 ablation in differentiated adipocytes, suggesting that Cx43 in adipocyte progenitors or other targeted cells restrains energy expenditures and promotes fat accumulation. These results reveal what we believe is a hitherto unknown action of Cx43 in adiposity, and offer a promising new pharmacologic target for improving metabolic balance in diabetes and obesity.

Connexin43 in mesenchymal lineage cells regulates body adiposity and energy metabolism in mice.

Connexin43 (Cx43) is the most abundant gap junction protein present in the mesenchymal lineage. In mature adipocytes, Cx43 mediates white adipose tissue (WAT) "beiging" in response to cold exposure and maintains the mitochondrial integrity of brown adipose tissue (BAT). We found that genetic deletion of Gja1 (Cx43 gene) in cells that give rise to chondro-osteogenic and adipogenic precursors driven by the Dermo1/Twist2 promoter leads to lower body adiposity and partial protection against the weight gain and metabolic syndrome induced by a high fat diet (HFD) in both sexes. These protective effects from obesogenic diet are related to increased locomotion, fuel utilization, energy expenditure, non-shivering thermogenesis, and better glucose tolerance in conditionally Gja1 ablated mice. Accordingly, Gja1 mutant mice exhibit reduced adipocyte hypertrophy, partially preserved insulin sensitivity, increased BAT lipolysis and decreased whitening under HFD. This metabolic phenotype is not reproduced with more restricted Gja1 ablation in differentiated adipocytes, suggesting that Cx43 has a hitherto unknown function in adipocyte progenitors or other targeted cells, resulting in restrained energy expenditures and fat accumulation. These results disclose an hitherto unknown action of Cx43 in adiposity, and offer a promising new pharmacologic target for improving metabolic balance in diabetes and obesity.

Systemic Activation of Activin A Signaling Causes Chronic Kidney Disease-Mineral Bone Disorder.

The high cardiovascular mortality associated with chronic kidney disease (CKD) is caused in part by the CKD-mineral bone disorder (CKD-MBD) syndrome. The CKD-MBD consists of skeletal, vascular and cardiac pathology caused by metabolic derangements produced by kidney disease. The prevalence of osteopenia/osteoporosis resulting from the skeletal component of the CKD-MBD, renal osteodystrophy (ROD), in patients with CKD exceeds that of the general population and is a major public health concern. That CKD is associated with compromised bone health is widely accepted, yet the mechanisms underlying impaired bone metabolism in CKD are not fully understood. Therefore, clarification of the molecular mechanisms by which CKD produces ROD is of crucial significance. We have shown that activin A, a member of the transforming growth factor (TGF)-ß super family, is an important positive regulator of receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis with Smad-mediated signaling being crucial for inducing osteoclast development and function. Recently, we have demonstrated systemic activation of activin receptors and activin A levels in CKD mouse models, such as diabetic CKD and Alport (AL) syndrome. In these CKD mouse models, bone remodeling caused by increased osteoclast numbers and activated osteoclastic bone resorption was observed and treatment with an activin receptor ligand trap repaired CKD-induced-osteoclastic bone resorption and stimulated individual osteoblastic bone formation, irrespective of parathyroid hormone (PTH) elevation. These findings have opened a new field for exploring mechanisms of activin A-enhanced osteoclast formation and function in CKD. Activin A appears to be a strong candidate for CKD-induced high-turnover ROD. Therefore, the treatment with the decoy receptor for activin A might be a good candidate for treatment for CKD-induced osteopenia or osteoporosis, indicating that the new findings from in these studies will lead to the identification of novel therapeutic targets for CKD-related and osteopenia and osteoporosis in general. In this review, we describe the impact of CKD-induced Smad signaling in osteoclasts, osteoblasts and vascular cells in CKD.

The activin receptor is stimulated in the skeleton, vasculature, heart, and kidney during chronic kidney disease.

We examined activin receptor type IIA (ActRIIA) activation in chronic kidney disease (CKD) by signal analysis and inhibition in mice with Alport syndrome using the ActRIIA ligand trap RAP-011 initiated in 75-day-old Alport mice. At 200 days of age, there was severe CKD and associated Mineral and Bone Disorder (CKD-MBD), consisting of osteodystrophy, vascular calcification, cardiac hypertrophy, hyperphosphatemia, hyperparathyroidism, elevated FGF23, and reduced klotho. The CKD-induced bone resorption and osteoblast dysfunction was reversed, and bone formation was increased by RAP-011. ActRIIA inhibition prevented the formation of calcium apatite deposits in the aortic adventitia and tunica media and significantly decreased the mean aortic calcium concentration from 0.59 in untreated to 0.36 mg/g in treated Alport mice. Aortic ActRIIA stimulation in untreated mice increased p-Smad2 levels and the transcription of sm22α and αSMA. ActRIIA inhibition reversed aortic expression of the osteoblast transition markers Runx2 and osterix. Heart weight was significantly increased by 26% in untreated mice but remained normal during RAP-011 treatment. In 150-day-old mice, GFR was significantly reduced by 55%, but only by 30% in the RAP-011-treated group. In 200-day-old mice, the mean BUN was 100 mg/dl in untreated mice compared to 60 mg/dl in the treated group. In the kidneys of 200-day-old mice, ActRIIA and p-Smad2 were induced and MCP-1, fibronectin, and interstitial fibrosis were stimulated; all were attenuated by RAP-011 treatment. Hence, the activation of ActRIIA signaling during early CKD contributes to the CKD-MBD components of osteodystrophy and cardiovascular disease and to renal fibrosis. Thus, the inhibition of ActRIIA signaling is efficacious in improving and delaying CKD-MBD in this model of Alport syndrome.

The chronic kidney disease - Mineral bone disorder (CKD-MBD): Advances in pathophysiology.

The causes of excess cardiovascular mortality associated with chronic kidney disease (CKD) have been attributed in part to the CKD-mineral bone disorder syndrome (CKD-MBD), wherein, novel cardiovascular risk factors have been identified. New advances in the causes of the CKD-MBD are discussed in this review. They demonstrate that repair and disease processes in the kidneys release factors to the circulation that cause the systemic complications of CKD. The discovery of WNT inhibitors, especially Dickkopf 1 (Dkk1), produced during renal repair as participating in the pathogenesis of the vascular and skeletal components of the CKD-MBD implied that additional pathogenic factors are critical. This lead to the discovery that activin A is a second renal repair factor circulating in increased levels during CKD. Activin A derives from peritubular myofibroblasts of diseased kidneys, wherein it stimulates fibrosis, and decreases tubular klotho expression. Activin A binds to the type 2 activin A receptor, ActRIIA, which is variably affected by CKD in the vasculature. In diabetic/atherosclerotic aortas, specifically in vascular smooth muscle cells (VSMC), ActRIIA signaling is inhibited and contributes to CKD induced VSMC dedifferentiation, osteogenic transition and neointimal atherosclerotic calcification. In nondiabetic/nonatherosclerotic aortas, CKD increases VSMC ActRIIA signaling, and vascular fibroblast signaling causing the latter to undergo osteogenic transition and stimulate vascular calcification. In both vascular situations, a ligand trap for ActRIIA prevented vascular calcification. In the skeleton, activin A is responsible for CKD stimulation of osteoclastogenesis and bone remodeling increasing bone turnover. These studies demonstrate that circulating renal repair and injury factors are causal of the CKD-MBD and CKD associated cardiovascular disease.

Ligand trap of the activin receptor type IIA inhibits osteoclast stimulation of bone remodeling in diabetic mice with chronic kidney disease.

Dysregulation of skeletal remodeling is a component of renal osteodystrophy. Previously, we showed that activin receptor signaling is differentially affected in various tissues in chronic kidney disease (CKD). We tested whether a ligand trap for the activin receptor type 2A (RAP-011) is an effective treatment of the osteodystrophy of the CKD-mineral bone disorder. With a 70% reduction in the glomerular filtration rate, CKD was induced at 14 weeks of age in the ldlr-/- high fat-fed mouse model of atherosclerotic vascular calcification and diabetes. Twenty mice with CKD, hyperphosphatemia, hyperparathyroidism, and elevated activin A were treated with RAP-011, wherease 19 mice were given vehicle twice weekly from week 22 until the mice were killed at 28 weeks of age. The animals were then evaluated by skeletal histomorphometry, micro-computed tomography, mechanical strength testing, and ex vivo bone cell culture. Results in the CKD groups were compared with those of the 16 sham-operated ldlr-/- high fat-fed mice. Sham-operated mice had low-turnover osteodystrophy and skeletal frailty. CKD stimulated bone remodeling with significant increases in osteoclast and osteoblast numbers and bone resorption. Compared with mice with CKD and sham-operated mice, RAP-011 treatment eliminated the CKD-induced increase in these histomorphometric parameters and increased trabecular bone fraction. RAP-011 significantly increased cortical bone area and thickness. Activin A-enhanced osteoclastogenesis was mediated through p-Smad2 association with c-fos and activation of nuclear factor of activated T cells c1 (NFATc1). Thus, an ActRIIA ligand trap reversed CKD-stimulated bone remodeling, likely through inhibition of activin-A induced osteoclastogenesis.

Ligand trap for the activin type IIA receptor protects against vascular disease and renal fibrosis in mice with chronic kidney disease.

The causes of cardiovascular mortality associated with chronic kidney disease (CKD) are partly attributed to the CKD-mineral bone disorder (CKD-MBD). The causes of the early CKD-MBD are not well known. Our discovery of Wnt (portmanteau of wingless and int) inhibitors, especially Dickkopf 1, produced during renal repair as participating in the pathogenesis of the vascular and skeletal components of the CKD-MBD implied that additional pathogenic factors are critical. In the search for such factors, we studied the effects of activin receptor type IIA (ActRIIA) signaling by using a ligand trap for the receptor, RAP-011 (a soluble extracellular domain of ActRIIA fused to a murine IgG-Fc fragment). In a mouse model of CKD that stimulated atherosclerotic calcification, RAP-011 significantly increased aortic ActRIIA signaling assessed by the levels of phosphorylated Smad2/3. Furthermore, RAP-011 treatment significantly reversed CKD-induced vascular smooth muscle dedifferentiation as assessed by smooth muscle 22α levels, osteoblastic transition, and neointimal plaque calcification. In the diseased kidneys, RAP-011 significantly stimulated αklotho levels and it inhibited ActRIIA signaling and decreased renal fibrosis and proteinuria. RAP-011 treatment significantly decreased both renal and circulating Dickkopf 1 levels, showing that Wnt activation was downstream of ActRIIA. Thus, ActRIIA signaling in CKD contributes to the CKD-MBD and renal fibrosis. ActRIIA signaling may be a potential therapeutic target in CKD.

SIRT6 deficiency culminates in low-turnover osteopenia.

Deficiency of Sirtuin 6 (SIRT6), a chromatin-related deacetylase, in mice reveals severe premature aging phenotypes including osteopenia. However, the underlying molecular mechanisms of SIRT6 in bone metabolism are unknown. Here we show that SIRT6 deficiency in mice produces low-turnover osteopenia caused by impaired bone formation and bone resorption, which are mechanisms similar to those of age-related bone loss. Mechanistically, SIRT6 interacts with runt-related transcription factor 2 (Runx2) and osterix (Osx), which are the two key transcriptional regulators of osteoblastogenesis, and deacetylates histone H3 at Lysine 9 (H3K9) at their promoters. Hence, excessively elevated Runx2 and Osx in SIRT6(-/-) osteoblasts lead to impaired osteoblastogenesis. In addition, SIRT6 deficiency produces hyperacetylation of H3K9 in the promoter of dickkopf-related protein 1 (Dkk1), a potent negative regulator of osteoblastogenesis, and osteoprotegerin, an inhibitor of osteoclastogenesis. Therefore, the resulting up-regulation of Dkk1 and osteoprotegerin levels contribute to impaired bone remodeling, leading to osteopenia with a low bone turnover in SIRT6-deficient mice. These results establish a new link between SIRT6 and bone remodeling that positively regulates osteoblastogenesis and osteoclastogenesis.

Pathophysiology of the chronic kidney disease-mineral bone disorder.

PURPOSE OF REVIEW: The causes of excess cardiovascular mortality associated with chronic kidney disease (CKD) have been attributed in part to the CKD-mineral bone disorder syndrome (CKD-MBD), wherein, novel cardiovascular risk factors have been identified. The causes of the CKD-MBD are not well known and they will be discussed in this review RECENT FINDINGS: The discovery of WNT (portmanteau of wingless and int) inhibitors, especially Dickkopf 1, produced during renal repair and participating in the pathogenesis of the vascular and skeletal components of the CKD-MBD implied that additional pathogenic factors are critical, leading to the finding that activin A is a second renal repair factor circulating in increased levels during CKD. Activin A derives from peritubular myofibroblasts of diseased kidneys, where it stimulates fibrosis, and decreases tubular klotho expression. The type 2 activin A receptor, ActRIIA, is decreased by CKD in atherosclerotic aortas, specifically in vascular smooth muscle cells (VSMC). Inhibition of activin signaling by a ligand trap inhibited CKD induced VSMC dedifferentiation, osteogenic transition and atherosclerotic calcification. Inhibition of activin signaling in the kidney decreased renal fibrosis and proteinuria. SUMMARY: These studies demonstrate that circulating renal repair factors are causal for the CKD-MBD and CKD associated cardiovascular disease, and identify ActRIIA signaling as a therapeutic target in CKD that links progression of renal disease and vascular disease.

CKD-induced wingless/integration1 inhibitors and phosphorus cause the CKD-mineral and bone disorder.

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-α, 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.

Expression of DGCR8-dependent microRNAs is indispensable for osteoclastic development and bone-resorbing activity.

Recently, microRNAs (miRs) have been implicated in bone formation and homeostasis. We previously reported that Dicer generated miRs have pivotal roles in differentiation and activity of osteoclasts. However, recent studies have demonstrated that Dicer is implicated in production of endogenous small interfering RNAs, non-canonical miRs, and other small RNAs in mammals. Hence, a challenging question is the extent to which expression of canonical miRs is obligatory for osteoclastic control of bone metabolism. DiGeorge syndrome critical region gene 8 (DGCR8) is exclusively related to expression of miRs by a canonical processing pathway together with the nuclear RNase III enzyme Drosha. Osteoclast-specific deletion of DGCR8 led to impaired osteoclastic development and bone resorption so that bone development was significantly retarded. In culture, the expression levels of osteoclastic phenotype-related genes and proteins were remarkably inhibited during osteoclastogenesis in DGCR8-deficiency. Thus, we have identified that DGCR8-dependent miRs are indispensable for osteoclastic control of bone metabolism.

Early chronic kidney disease-mineral bone disorder stimulates vascular calcification.

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.

Down-regulation of miR-21 biogenesis by estrogen action contributes to osteoclastic apoptosis.

Estrogen inhibits osteoclastogenesis and induces osteoclastic apoptosis; however, the molecular mechanisms remain controversial. Recently, a group has demonstrated that osteoclasts are a direct target for estrogen because estrogen stimulates transcription of the Fas Ligand (FasL) gene in osteoclasts, which in turn causes cell death through an autocrine mechanism. In contrast, other groups have shown that the cells are an indirect target for estrogen because estrogen fails to stimulate the transcription of that in osteoclasts. Thus, two quite different molecular mechanisms have been suggested to explain the effects of estrogen in osteoclastic apoptosis. Here we show that the proapoptotic effect of estrogen during osteoclastogenesis is regulated by a posttranscriptional increase in FasL production by down-regulated microRNA-21 (miR-21) biogenesis. Previously, we reported that miR-21 is highly expressed in osteoclastogenesis. We found that estrogen down-regulates miR-21 biogenesis so that FasL, the targets of miR-21, protein levels are posttranscriptionally increased that induce osteoclastic apoptosis. Moreover, the gain-of-function of miR-21 rescued the apoptosis. In addition, we failed to detect estrogen-enhanced FasL levels at mRNA levels. Thus, osteoclastic survival is controlled by autocrine actions of FasL regulated by estrogen and miR-21 plays a central role during estrogen-controlled osteoclastogenesis.

Cardiovascular risk factors in chronic kidney disease: does phosphate qualify?

Risk factors for disease states are rigorously defined. This analysis considers the definition of a risk factor as applied to the question of whether the serum phosphorus level is a risk factor for cardiovascular disease. Observational studies strongly suggest that phosphorus is associated with cardiovascular risk, and definitive prospective animal studies are supportive. A plausible mechanism of action has been discovered demonstrating that phosphorus stimulates osteoblastic transition of cells in the neointima of atherosclerotic plaques, which, if prevented, blocks vascular calcification. However, prospective studies demonstrating that modulation of the putative risk factor affects clinical outcomes are lacking, and phosphorus, as yet, does not qualify as a cardiovascular risk factor. This is a clarion call for additional research.

A microRNA expression signature of osteoclastogenesis.

MicroRNAs (miRs) are small noncoding RNAs that principally function in the spatiotemporal regulation of protein translation in animal cells. Although emerging evidence suggests that some miRs play important roles in osteoblastogenesis and skeletal homeostasis, much less is known in osteoclastogenesis. Here, we show that receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis is mediated by miR-21. MiR-21 was identified as an miR expression signature of RANKL-induced osteoclastogenesis that down-regulates programmed cell death 4 (PDCD4) protein levels. Diminished PDCD4 removes a repression from c-Fos, a critical transcription factor for osteoclastogenesis and osteoclast-specific downstream target genes. In addition, RANKL-induced c-Fos up-regulates miR-21 gene expression. Bone marrow-derived monocyte/macrophage precursors deficient of DiGeorge syndrome critical region gene 8, an RNA binding protein associated with miR biogenesis, and Dicer, an endoribonuclease in the RNaseIII family associated with miR biogenesis, possessed significantly decreased miR-21 levels and increased PDCD4 protein levels so that RANKL-induced osteoclastogenesis was impaired in those cells. However, forced expression of miR-21 rescued osteoclast development because of down-regulation of PDCD4 protein expression levels. Thus, our studies provide a new molecular mechanism, including a positive feedback loop of c-Fos/miR-21/PDCD4, regulating osteoclastogenesis.

Cardiovascular risk factors in chronic kidney disease: does phosphate qualify?

Risk factors for disease states are rigorously defined. This analysis considers the definition of a risk factor as applied to the question of whether the serum phosphorus level is a risk factor for cardiovascular disease. Observational studies strongly suggest that phosphorus is associated with cardiovascular risk, and definitive prospective animal studies are supportive. A plausible mechanism of action has been discovered demonstrating that phosphorus stimulates osteoblastic transition of cells in the neointima of atherosclerotic plaques, which, if prevented, blocks vascular calcification. However, prospective studies demonstrating that modulation of the putative risk factor affects clinical outcomes are lacking, and phosphorus, as yet, does not qualify as a cardiovascular risk factor. This is a clarion call for additional research.

Impaired micro-RNA pathways diminish osteoclast differentiation and function.

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.

The mechanism of phosphorus as a cardiovascular risk factor in CKD.

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.

Akt1/Akt2 and mammalian target of rapamycin/Bim play critical roles in osteoclast differentiation and survival, respectively, whereas Akt is dispensable for cell survival in isolated osteoclast precursors.

Akt, also known as protein kinase B, is a serine/threonine protein kinase with antiapoptotic activities; also, it is a downstream target of phosphatidylinositol 3-kinase. Here we show that Akt1/Akt2 play a critical role in osteoclast differentiation but not cell survival and that mammalian target of rapamycin (mTOR) and Bim, a pro-apoptotic Bcl-2 family member, are required for cell survival in isolated osteoclast precursors. To investigate the function of Akt1, Akt2, mTOR, and Bim, we employed a retroviral system for delivery of small interfering RNA into cells. Loss of Akt1 and/or Akt2 protein inhibited osteoclast differentiation due to down-regulation of IkappaB-kinase (IKK) alpha/beta activity, phosphorylation of IkappaB-alpha, nuclear translocation of nuclear factor-kappaB (NFkappaB) p50, and NFkappaB p50 DNA-binding activity. Surprisingly, deletion of Akt1 and/or Akt2 protein did not stimulate cleaved caspase-3 activity and failed to promote apoptosis. Conversely, loss of mTOR protein induced apoptosis due to up-regulation of cleaved caspase-3 activity. In addition, we found that mTOR is downstream of phosphatidylinositol 3-kinase (but not Akt) and that macrophage colony-stimulating factor regulates Bim expression through mTOR activation for cell survival. These results demonstrate that Akt1/Akt2 are key elements in osteoclast differentiation and that the macrophage colony-stimulating factor stimulation of mTOR leading to Bim inhibition is essential for cell survival in isolated osteoclast precursors.

Effect of the phosphodiesterase 4 inhibitor, rolipram, on retinoic acid-increased alkaline phosphatase activity in the mouse fibroblastic C3H10T1/2 cell line.

We have evaluated effects of a phosphodiesterase (PDE) 4 inhibitor on retinoic acid-increased alkaline phosphatase activity in the mouse fibroblastic C3H10T1/2 clone 8 (10T1/2) cell line. 10T1/2 cells were cultured in minimum essential medium (MEM) and 10% fetal bovine serum with or without 1 microM retinoic acid and/or the PDE 4 inhibitor, rolipram, and harvested at specific intervals before measurement of alkaline phosphatase activity, cAMP production in response to parathyroid hormone, osteocalcin synthesis and expression, and phosphodiesterase activity. Retinoic acid-increased alkaline phosphatase activity, and slightly enhanced cAMP production in response to parathyroid hormone. However, it did not affect osteocalcin synthesis and expression. In the presence of retinoic acid, PDE 4 activity was not changed. A PDE 4 inhibitor, rolipram, and cAMP analog, 8-bromo-cAMP dramatically increased retinoic acid's ability to induce alkaline phosphatase activity. This is the first report that PDE 4 may be involved in regulation of retinoic acid-increased alkaline phosphatase activity.