Rebecca Berdeaux

SIK1 is a class II HDAC kinase that promotes survival of skeletal myocytes

During physical exercise, increases in motor neuron activity stimulate the expression of muscle-specific genes through the myocyte enhancer factor 2 (MEF2) family of transcription factors. Elevations in intracellular calcium increase MEF2 activity via the phosphorylation-dependent inactivation of class II histone deacetylases (HDACs). In studies to determine the role of the cAMP responsive element binding protein (CREB) in skeletal muscle, we found that mice expressing a dominant-negative CREB transgene (M-ACREB mice) exhibited a dystrophic phenotype along with reduced MEF2 activity. Class II HDAC phosphorylation was decreased in M-ACREB myofibers due to a reduction in amounts of Snf1lk (encoding salt inducible kinase, SIK1), a CREB target gene that functions as a class II HDAC kinase. Inhibiting class II HDAC activity either by viral expression of Snf1lk or by the administration of a small molecule antagonist improved the dystrophic phenotype in M-ACREB mice, pointing to an important role for the SIK1-HDAC pathway in regulating muscle function.

Active Rho is localized to podosomes induced by oncogenic Src and is required for their assembly and function.

Transformation of fibroblasts by oncogenic Src causes disruption of actin stress fibers and formation of invasive adhesions called podosomes. Because the small GTPase Rho stimulates stress fiber formation, Rho inactivation by Src has been thought to be necessary for stress fiber disruption. However, we show here that Rho[GTP] levels do not decrease after transformation by activated Src. Inactivation of Rho in Src-transformed fibroblasts by dominant negative RhoA or the Rho-specific inhibitor C3 exoenzyme disrupted podosome structure as judged by localization of podosome components F-actin, cortactin, and Fish. Inhibition of Rho strongly inhibited Src-induced proteolytic degradation of the extracellular matrix. Furthermore, development of an in situ Rho[GTP] affinity assay allowed us to detect endogenous Rho[GTP] at podosomes, where it colocalized with F-actin, cortactin, and Fish. Therefore, Rho is not globally inactivated in Src-transformed fibroblasts, but is necessary for the assembly and function of structures implicated in tumor cell invasion.

Dual role of SnoN in mammalian tumorigenesis

ABSTRACT SnoN is an important negative regulator of transforming growth factor β signaling through its ability to interact with and repress the activity of Smad proteins. It was originally identified as an oncoprotein based on its ability to induce anchorage-independent growth in chicken embryo fibroblasts. However, the roles of SnoN in mammalian epithelial carcinogenesis have not been well defined. Here we show for the first time that SnoN plays an important but complex role in human cancer. SnoN expression is highly elevated in many human cancer cell lines, and this high level of SnoN promotes mitogenic transformation of breast and lung cancer cell lines in vitro and tumor growth in vivo, consistent with its proposed prooncogenic role. However, this high level of SnoN expression also inhibits epithelial-to-mesenchymal transdifferentiation. Breast and lung cancer cells expressing the shRNA for SnoN exhibited an increase in cell motility, actin stress fiber formation, metalloprotease activity, and extracellular matrix production as well as a reduction in adherens junction proteins. Supporting this observation, in an in vivo breast cancer metastasis model, reducing SnoN expression was found to moderately enhance metastasis of human breast cancer cells to bone and lung. Thus, SnoN plays both protumorigenic and antitumorigenic roles at different stages of mammalian malignant progression. The growth-promoting activity of SnoN appears to require its ability to bind to and repress the Smad proteins, while the antitumorigenic activity can be mediated by both Smad-dependent and Smad-independent pathways and requires the activity of small GTPase RhoA. Our study has established the importance of SnoN in mammalian epithelial carcinogenesis and revealed a novel aspect of SnoN function in malignant progression.

CRTC3 links catecholamine signalling to energy balance

The adipose-derived hormone leptin maintains energy balance in part through central nervous system-mediated increases in sympathetic outflow that enhance fat burning. Triggering of β-adrenergic receptors in adipocytes stimulates energy expenditure by cyclic AMP (cAMP)-dependent increases in lipolysis and fatty-acid oxidation. Although the mechanism is unclear, catecholamine signalling is thought to be disrupted in obesity, leading to the development of insulin resistance. Here we show that the cAMP response element binding (CREB) coactivator Crtc3 promotes obesity by attenuating β-adrenergic receptor signalling in adipose tissue. Crtc3 was activated in response to catecholamine signals, when it reduced adenyl cyclase activity by upregulating the expression of Rgs2, a GTPase-activating protein that also inhibits adenyl cyclase activity. As a common human CRTC3 variant with increased transcriptional activity is associated with adiposity in two distinct Mexican-American cohorts, these results suggest that adipocyte CRTC3 may play a role in the development of obesity in humans.

Ras-independent activation of Ral by a Ca2+-dependent pathway

The RalA and RalB proteins comprise a distinct family of small GTPases [1]. Ral-specific guanine-nucleotide exchange factors such as RalGDS, Rlf and RGL interact with activated Ras and cooperate with Ras in the transformation of murine fibroblasts [2-5]. Thus, the interaction of RalGDS with Ras and the subsequent activation of Ral are thought to constitute a distinct Ras-dependent signaling pathway. The function of Ral is largely unknown. There is circumstantial evidence that Ral may have a function in regulating the cytoskeleton through its interaction with RIP1 (also known as RLIP or RalBP1), a GTPase-activating protein specific for the small GTPases Cdc42 and Rac [6-8]. Ral also binds to phospholipase D (PLD) and thus may play a role in signaling through phospholipids [9]. We have examined endogenous levels of activated, GTP-bound Ral (Ral-GTP) in Rat-2 fibroblasts stimulated with various mitogens. Lysophosphatidic acid (LPA) and epidermal growth factor (EGF), which activate both Ras-dependent and Ras-independent signaling pathways [10,11], rapidly activated Ral. Inhibition of Ras activation by dominant-negative Ras (RasS17N) or pertussis toxin had little effect on Ral-GTP levels, however. Ral was activated by the Ca2+ ionophore ionomycin, and activation by LPA or EGF could be blocked by a phospholipase C (PLC) inhibitor. The results presented here demonstrate a Ca(2+)-dependent mechanism for the activation of Ral.

The effects of obesity on skeletal muscle regeneration

Obesity and metabolic disorders such as type 2 diabetes mellitus are accompanied by increased lipid deposition in adipose and non-adipose tissues including liver, pancreas, heart and skeletal muscle. Recent publications report impaired regenerative capacity of skeletal muscle following injury in obese mice. Although muscle regeneration has not been thoroughly studied in obese and type 2 diabetic humans and mechanisms leading to decreased muscle regeneration in obesity remain elusive, the initial findings point to the possibility that muscle satellite cell function is compromised under conditions of lipid overload. Elevated toxic lipid metabolites and increased pro-inflammatory cytokines as well as insulin and leptin resistance that occur in obese animals may contribute to decreased regenerative capacity of skeletal muscle. In addition, obesity-associated alterations in the metabolic state of skeletal muscle fibers and satellite cells may directly impair the potential for satellite cell-mediated repair. Here we discuss recent studies that expand our understanding of how obesity negatively impacts skeletal muscle maintenance and regeneration.

MEF2 is an in vivo immune-metabolic switch.

Summary Infections disturb metabolic homeostasis in many contexts, but the underlying connections are not completely understood. To address this, we use paired genetic and computational screens in Drosophila to identify transcriptional regulators of immunity and pathology and their associated target genes and physiologies. We show that Mef2 is required in the fat body for anabolic function and the immune response. Using genetic and biochemical approaches, we find that MEF2 is phosphorylated at a conserved site in healthy flies and promotes expression of lipogenic and glycogenic enzymes. Upon infection, this phosphorylation is lost, and the activity of MEF2 changes—MEF2 now associates with the TATA binding protein to bind a distinct TATA box sequence and promote antimicrobial peptide expression. The loss of phosphorylated MEF2 contributes to loss of anabolic enzyme expression in Gram-negative bacterial infection. MEF2 is thus a critical transcriptional switch in the adult fat body between metabolism and immunity.

Angiotensin II type-2 (AT2) receptor-mediated inhibition of NMDA receptor signalling in neuronal cells

The N-methyl-D-aspartate (NMDA) receptor has been reported to be important in synaptic plasticity, neuronal development, normal brain function and neurologic disease. We have recently shown that PC12W cells, a subclone of rat pheochromocytoma PC12 cell line, release nitric oxide (NO), as measured by in vitro spin-trapping combined with electron paramagnetic resonance (EPR) spectroscopy, when challenged with NMDA [Norby, S.W., Weyhenmeyer, J.A. and Clarkson, R.B., Stimulation and inhibition of NO production in macrophages and neuronal cells as observed by spin trapping, Free Rad. Biol. Med., 22 (1997) 1-9]. In the present study, we provide immunochemical evidence for the expression of both the NMDAR1 and NMDAR2A/B receptor subunits in PC12W cells, that express only the angiotensin type-2 (AT2) receptor subtype, and in NG108-15 (NG108) cells, a murine neuroblastoma x glioma hybrid that expresses both the angiotensin type-1 (AT1) and AT2 receptor subtypes. We also show that treatment of PC12W cells with angiotensin (Ang II) decreases NMDA-induced NO release by 28.0 +/- 4.2%, and that this response can be attenuated by pre-treating the cells with the isoform-specific AT2 antagonist, PD 123319. Interestingly, there was no effect on cGMP accumulation in PC12W cells treated with NMDA. Similar experiments were carried out using NG108 cells since the binding properties and functional characteristics of their NMDA receptors have been previously described [Ohkuma, S., Katsura, M., Chen, D., Chen, S. and Kuriyama, K., Presence of N-methyl-D-aspartate (NMDA) receptors in neuroblastoma x glioma hybrid NG 108-15 cells-analysis using 45Ca2+ influx and [3H]MK-801 binding as functional measures, Mol. Brain Res. 22 (1994) 166-172]. Our results show that NG108 cells significantly increase cGMP levels when challenged with NMDA (21.2 +/- 5.0% over control levels), and that this response can be attenuated by the addition of angiotensin (57.1 +/- 6.2% of stimulated levels). The effect of angiotensin on NMDA-mediated changes in cGMP levels was blocked by the AT2 antagonist, PD 123319, but was not significantly changed by the addition of the AT1 antagonist, losartan. Further, Ang II action on NMDA signalling in NG108 cells was completely inhibited by the addition of both the AT1 and AT2 antagonists. Taken together, these results suggest that AngII inhibits NMDA-mediated NO and cGMP production through a mechanism involving the AT2 receptor subtype.

Brain and muscle Arnt-like 1 is a key regulator of myogenesis

Summary The circadian clock network is an evolutionarily conserved mechanism that imparts temporal regulation to diverse biological processes. Brain and muscle Arnt-like 1 (Bmal1), an essential transcriptional activator of the clock, is highly expressed in skeletal muscle. However, whether this key clock component impacts myogenesis, a temporally regulated event that requires the sequential activation of myogenic regulatory factors, is not known. Here we report a novel function of Bmal1 in controlling myogenic differentiation through direct transcriptional activation of components of the canonical Wnt signaling cascade, a major inductive signal for embryonic and postnatal muscle growth. Genetic loss of Bmal1 in mice leads to reduced total muscle mass and Bmal1-deficient primary myoblasts exhibit significantly impaired myogenic differentiation accompanied by markedly blunted expression of key myogenic regulatory factors. Conversely, forced expression of Bmal1 enhances differentiation of C2C12 myoblasts. This cell-autonomous effect of Bmal1 is mediated by Wnt signaling as both expression and activity of Wnt components are markedly attenuated by inhibition of Bmal1, and activation of the Wnt pathway partially rescues the myogenic defect in Bmal1-deficient myoblasts. We further reveal direct association of Bmal1 with promoters of canonical Wnt pathway genes, and as a result of this transcriptional regulation, Wnt signaling components exhibit intrinsic circadian oscillation. Collectively, our study demonstrates that the core clock gene, Bmal1, is a positive regulator of myogenesis, which may represent a temporal regulatory mechanism to fine-tune myocyte differentiation.

Creb is activated by muscle injury and promotes muscle regeneration

The cAMP response element binding protein (CREB) plays key roles in differentiation of embryonic skeletal muscle progenitors and survival of adult skeletal muscle. However, little is known about the physiologic signals that activate CREB in normal muscle. Here we show that CREB phosphorylation and target genes are induced after acute muscle injury and during regeneration due to genetic mutation. Activated CREB localizes to both myogenic precursor cells and newly regenerating myofibers within regenerating areas. Moreover, we found that signals from damaged skeletal muscle tissue induce CREB phosphorylation and target gene expression in primary mouse myoblasts. An activated CREB mutant (CREBY134F) potentiates myoblast proliferation as well as expression of early myogenic transcription factors in cultured primary myocytes. Consistently, activated CREB-YF promotes myoblast proliferation after acute muscle injury in vivo and enhances muscle regeneration in dystrophic mdx mice. Our findings reveal a new physiologic function for CREB in contributing to skeletal muscle regeneration.