SUMMARY Bone formation in extant species is restricted to vertebrate species. Sp7/Osterix is a key transcriptional determinant of bone-secreting osteoblasts. We performed Sp7 ChIP-seq analysis identifying a large set of predicted osteoblast enhancers and validated a subset of these in cell culture and transgenic mouse assays. Sp-family members bind GC-rich target sequences through their zinc finger domain. Several lines of evidence suggest Sp7 acts differently, engaging osteoblast targets in Dlx-containing regulatory complexes bound to AT-rich motifs. Amino acid differences in the Sp7 zinc finger domain reduce Sp7's affinity for the Sp-family consensus GC-box target; Dlx5 binding maps to this domain of Sp7. The data support a model in which Dlx recruitment of Sp7 to osteoblast enhancers underlies Sp7-directed osteoblast specification. As an Sp7-like zinc finger variant is restricted to vertebrates, the emergence of an Sp7 member within the Sp family was likely closely coupled to evolution of the bone forming vertebrates.

Background For reasons that remain unclear, whether type 5 AC (AC5), one of two major AC isoforms in heart, is protective or deleterious in response to cardiac stress is controversial. To reconcile this controversy we examined the cardiomyopathy induced by chronic isoproterenol (ISO) in AC5 transgenic (Tg) mice and the signaling mechanisms involved. Methods and Results Chronic ISO increased oxidative stress and induced more severe cardiomyopathy in AC5 Tg, as left ventricular (LV) ejection fraction fell 1.9 fold more than wild type (WT), along with greater LV dilation and increased fibrosis, apoptosis and hypertrophy. Oxidative stress induced by chronic ISO, detected by 8-OhDG was 15% greater, p=0.007, in AC5 Tg hearts, while protein expression of MnSOD was reduced by 38%, indicating that the susceptibility of AC5 Tg to cardiomyopathy may be due to decreased MnSOD expression. Consistent with this, susceptibility of the AC5 Tg to cardiomyopathy was suppressed by overexpression of MnSOD, whereas protection afforded by the AC5 KO was lost in AC5 KO×MnSOD+/− mice. Elevation of MnSOD was eliminated by both sirtuin and MEK inhibitors, suggesting both the SIRT1/FoxO3a and MEK/ERK pathway are involved in MnSOD regulation by AC5. Conclusion Overexpression of AC5 exacerbates the cardiomyopathy induced by chronic catecholamine stress by altering regulation of SIRT1/FoxO3a, MEK/ERK and MnSOD, resulting in oxidative stress intolerance, thereby shedding light on new approaches for treatment of heart failure.

Vatner SF, Ishikawa Y. Prevention of heart failure in mice by an antiviral agent that inhibits type 5 cardiac adenylyl cyclase. Am J Physiol Heart Circ Physiol 302: H2622-H2628, 2012. First published April 13, 2012; doi:10.1152/ajpheart.00190.2012.-Despite numerous discoveries from genetically engineered mice, relatively few have been translated to the bedside, mainly because it is difficult to translate from genes to drugs. This investigation examines an antiviral drug, which also has an action to selectively inhibit type 5 adenylyl cyclase (AC5), a pharmaceutical correlate of the AC5 knockout (KO) model, which exhibits longevity and stress resistance. Our objective was to examine the extent to which pretreatment with this drug, adenine 9-␤-D-arabinofuranoside (Ara-A), favorably ameliorates the development of heart failure (HF). Ara-A exhibited selective inhibition for AC5 compared with the other major cardiac AC isoform, AC6, i.e., it reduced AC activity significantly in AC5 transgenic (Tg) mice, but not in AC5KO mice and had little effect in either wild-type or AC6Tg mice. Permanent coronary artery occlusion for 3 wk in C57Bl/6 mice increased mortality and induced HF in survivors, as reflected by reduced cardiac function, while increasing cardiac fibrosis. The AC5 inhibitor Ara-A significantly improved all of these end points and also ameliorated chronic isoproterenol-induced cardiomyopathy. As with the AC5KO mice, Ara-A increased mitogen/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK) phosphorylation. A MEK inhibitor abolished the beneficial effects of the AC5 inhibitor in the HF model, indicating the involvement of the downstream MEK-ERK pathway of AC5. Our data suggest that pharmacological AC5 inhibition may serve as a new therapeutic approach for HF. heart failure; type 5 adenylyl cyclase inhibition; adenine 9-␤-Darabinofuranoside DESPITE GAINS IN THE TREATMENT of heart failure (HF) with both angiotensin and ␤-adrenergic receptor (␤-AR) blockers, HF still remains a major cause of death and disability. In addition, some patients do not tolerate ␤-AR blocking therapy (2). It is conceivable that inhibiting mechanisms distal to the ␤-AR signaling pathway, identified from genetically engineered mouse models, might be a novel approach. While there have been numerous potential therapeutic approaches discovered from studies in genetically engineered mice in the past two decades, there are relatively few of these discoveries that have been translated to the bedside, mainly because it is difficult to translate the effects of disrupting a gene in a mouse to therapy in patients with HF.The goal of this investigation was to examine the extent to which a pharmacological inhibitor of type 5 adenylyl cyclase (AC5), 9-␤-D-arabinofuranoside (Ara-A), could mimic the salutary effects observed in the AC5 knockout (KO) mice model, which protects against cardiac stress (10, 11) and increases longevity (15). The first goal was to determine the extent to which Ara-A selectively inhibits AC5...

Normal endochondral bone development requires the coordination of chondrocyte proliferation and differentiation. Indian hedgehog (Ihh) is a morphogen produced by chondrocytes in the early stage of terminal differentiation and plays several key roles in this process. Ihh regulates growth of adjacent proliferative chondrocytes and can also regulate the rate of differentiation of chondrocytes indirectly through its stimulation of parathyroid hormonerelated protein (PTHrP). In this review, we focus on recent studies that have identified new functions of Ihh and how Ihh itself is being regulated.

G protein-coupled receptor/adenylyl cyclase (AC)/cAMP signaling is crucial for all cellular responses to physiological and pathophysiological stimuli. There are nine isoforms of membrane-bound AC, with type 5 being one of the two major isoforms in the heart. Since the role of AC in the heart in regulating cAMP and acute changes in inotropic and chronotropic state are well known, this review will address our current understanding of the distinct regulatory role of the AC5 isoform in response to chronic stress. Transgenic overexpression of AC5 in cardiomyocytes of the heart (AC5-Tg) improves baseline cardiac function but impairs the ability of the heart to withstand stress. For example, chronic catecholamine stimulation induces cardiomyopathy, which is more severe in AC5-Tg mice, mediated through the AC5/sirtuin 1/forkhead box O3a pathway. Conversely, disrupting AC5, i.e., AC5 knockout, protects the heart from chronic catecholamine cardiomyopathy as well as the cardiomyopathies resulting from chronic pressure overload or aging. Moreover, AC5 knockout results in a 30% increase in a healthy life span, resembling the most widely studied model of longevity, i.e., calorie restriction. These two models of longevity share similar gene regulation in the heart, muscle, liver, and brain in that they are both protected against diabetes, obesity, and diabetic and aging cardiomyopathy. A pharmacological inhibitor of AC5 also provides protection against cardiac stress, diabetes, and obesity. Thus AC5 inhibition has novel, potential therapeutic applicability to several diseases not only in the heart but also in aging, diabetes, and obesity.

The sympathetic nervous system has been demonstrated to have a role in regulating bone remodeling through beta-adrenergic receptors (beta-AR) expressed on osteoblasts. Studies using beta(2)-adrenergic receptor agonists in vivo have also suggested an effect on endochondral bone development; however, it was not clear if this effect was mediated through osteoblasts or chondrocytes. To more thoroughly examine the role of beta-AR in chondrocytes we characterized the expression and signal transduction systems activated by beta-AR in growth plate chondrocytes prepared from ribs of embryonic E18.5 mice. Using RT-PCR and immunohistochemistry we found that the chondrocytes expressed only beta(2)-AR. The receptors were coupled to stimulation of adenylyl cyclase, phosphorylation of the cyclic AMP response element binding protein (CREB) and extracellular signal-regulated kinase (ERK1/2). Stimulation of ERK1/2 was transient and limited by the concomitant stimulation of the mitogen-activated protein kinase phosphatase (MKP-1). Isoproterenol stimulated the growth of chondrocytes as assessed by increased incorporation of [(3)H]-thymidine into the cells. The cellular expression of two markers of chondrocyte differentiation, Indian hedgehog, expressed in pre-hypertrophic cells and collagen type X, expressed in hypertrophic chondrocytes, were both significantly inhibited after incubation with isoproterenol. Collectively, these findings demonstrate regulation of chondrocytes through beta(2)-AR expressed on the cells that stimulate their growth and inhibit their differentiation, indicating that the sympathetic nervous system may be an important regulator of embryonic cartilage development.

Liver kinase b1 (Lkb1) protein kinase activity regulates cell growth and cell polarity. Here, we show Lkb1 is essential for maintaining a balance between mitotic and postmitotic cell fates in development of the mammalian skeleton. In this process, Lkb1 activity controls the progression of mitotic chondrocytes to a mature, postmitotic hypertrophic fate. Loss of this Lkb1-dependent switch leads to a dramatic expansion of immature chondrocytes and formation of enchondroma-like tumors. Pathway analysis points to a mammalian target of rapamycin complex 1-dependent mechanism that can be partially suppressed by rapamycin treatment. These findings highlight a critical requirement for integration of mammalian target of rapamycin activity into developmental decision-making during mammalian skeletogenesis.chondrocyte differentiation | endochondral ossification | cell death | hypoxia

The sympathetic nervous system can regulate both osteoblast and chondrocyte growth and activity through β(2)-adrenergic receptors (β(2)-AR). We have shown previously that β(2)-AR activate both adenylyl cyclase and mitogen-activated protein kinases ERK1/2 in growth plate chondrocytes prepared from ribs of embryonic E18.5 mice. Here we examined β(2)-AR inhibition of collagen type II (Col II) expression in growth plate chondrocytes and the molecular pathways involved. Stimulation of β(2)-AR by isoproterenol inhibited Col II mRNA and protein levels by ∼50% beginning at 2 h, with both remaining suppressed over 24 h. This inhibition was blocked by propranolol and inhibitors of either MEK1 or PKA. Isoproterenol stimulated an AP-1-luciferase reporter and increased the expression of AP-1 factors c-Fos, Fra-1, Fra-2, c-Jun, and Jun-B but had no effect on Jun-D. Stimulation of AP-1 activity was blocked by inhibitors of MEK1 or PKA. siRNA inhibition of AP-1 factors showed that depletion of only Jun-B attenuated isoproterenol-mediated inhibition of Col II. Transfection with jun-B or c-fos showed selective inhibition of Col II mRNA and a Col II luciferase reporter construct by jun-B. Isoproterenol as well as jun-B overexpression in the chondrocytes also inhibited the expression of Sox-6 mRNA and protein, and depletion of Jun-B abrogated β(2)-AR inhibition of Sox-6. Collectively, these findings demonstrate regulation of chondrocyte differentiation through β(2)-AR mediated by ERK1/2 and PKA stimulation of the AP-1 factor Jun-B that inhibits the expression of Sox-6 and Col II.