Browsing by Subject "Calcineurin"
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Item Control of Skeletal Muscle Fiber Types by Calcium Signaling Pathways(2002-08-01) Wu, Hai; William, R. SandersDifferent patterns of motor nerve activity drive distinctive programs of gene expression in skeletal muscles, thereby establishing a high degree of metabolic and physiological specialization among myofiber subtypes. Previous studies have demonstrated that calcineurin activity is required to maintain slow myofiber identity. I am interested in determining the transcription factors downstream of calcineurin and other calcium-regulated signaling pathways in the control of myofiber specialization. By analyzing two fiber type-specific enhancers, I was able to demonstrate that there are functional NFAT (nuclear factor of activated T cells) and MEF2 (myocyte-specific enhancer factor 2) binding sites within the enhancer of troponin I slow, and both sites are required for slow fiber specific activity of this enhancer. Next, I identified MEF2 as a target of calcineurin in cultured myogenic cells. Calcineurin physically interacts with MEF2 and dephosphorylates MEF2. C-terminal transactivation domain, but not N-terminal DNA binding domain of MEF2, responds to calcineurin activation. The use of "MEF2 indicator" transgenic mice that harbor a MEF2-dependent lacZ transgene enabled us to monitor the endogenous activities of MEF2 transcription factors. MEF2 is selectively active in slow and oxidative myofibers. Calcineurin is both necessary and sufficient for MEF2 activation in skeletal muscles. I also found a dose-response relationship between calcineurin activity and expression level of slow, oxidative fiber-specific and MEF2 target genes. Furthermore, I observed that functional activity of MEF2 transcription factors was stimulated by sustained periods of endurance exercise or low-frequency motor nerve pacing in a calcineurin-dependent manner. In addition to calcineurin, CaMKs (calcium, calmodulin-dependent kinases) also transduce their signaling through MEF2. CaMKIV synergistically activates MEF2-dependent gene expression together with calcineurin. Transgenic mice expressing constitutively active CaMKIV in their skeletal muscles showed increased percentage of slow and oxidative myofibers, which was accompanied by increased mitochondrial biogenesis mediated through the upregulation of PGC-1 (PPARg co-activator). Taken together, these findings delineate a molecular pathway in which MEF2 and NFAT integrate signaling inputs from multiple calcium-regulated pathways in the control of skeletal muscle fiber types.Item FHL2 Inhibits Calcineurin and Represses Pathological Cardiac Hypertrophy(2010-11-02) Hojayev, Berdymammet; Hill, Joseph A.Stress-induced cardiac hypertrophy is a hallmark feature of pathological remodeling which, left unchecked, predisposes hearts to arrhythmia and failure. FHL2 is a member of the four-and-a-half LIM domain (FHL) family of proteins expressed predominantly in the heart. Targeted disruption of FHL2 leads to an exaggerated response to beta-agonist (isoproterenol)-induced cardiac hypertrophy. Isoproterenol-induced hypertrophy relies on activation of the calcineurin-NFAT pathway, and inhibition of calcineurin is sufficient to block growth in response to isoproterenol. I also observed that FHL2 is up-regulated in mouse hearts after isoproterenol treatment. Based on this, we hypothesized that FHL2 negatively regulates the calcineurin-NFAT pathway and consequently, the hypertrophic growth response. To determine whether calcineurin signaling is enhanced in the absence of FHL2, wild type (WT) and FHL2 knockout (FHL2-/-) mice were treated with isoproterenol (32 mg/kg/day). We observed a significant increase in isoproterenol-induced expression of the NFAT target genes RCAN1.4 and BNP in FHL2-/- hearts as compared to WT. To determine whether the effect of FHL2 on the abundance of NFAT target gene transcripts was mediated by calcineurin-NFAT-dependent transcription, HEK 293 cells were transfected with luciferase reporter constructs containing the NFAT-driven promoters of either RCAN1 or IL-2. Consistent with the in vivo data, knockdown of FHL2 message using siRNA led to increases in both RCAN1 and IL-2 promoter activities elicited by constitutively active calcineurin or the calcium ionophore, ionomycin. Importantly, activation of the RCAN1 promoter by ionomycin, in control and FHL2 knockdown cells, was abolished by the calcineurin inhibitor cyclosporin A, confirming the calcineurin dependence of the response. Over-expression of FHL2 in HEK 293 cells inhibited the activation of both NFAT reporters triggered by either constitutively active calcineurin or ionomycin. Furthermore, neonatal rat ventricular myocytes over-expressing FHL2 exhibited reduced hypertrophic growth in response to constitutively active calcineurin (measured by cell cross-sectional area and fetal gene expression). Finally, immunostaining of adult cardiomyocytes revealed co-localization of FHL2 and calcineurin predominantly at the sarcomere, and activation of calcineurin by endothelin-1 treatment resulted in interaction between FHL2 and calcineurin as demonstrated by coimmunoprecipitation. These observations demonstrate that FHL2 represses calcineurin-NFAT signaling and thereby suppresses hypertrophic cardiac growth at least in part by interacting with calcineurin and inhibiting its activation.Item MAPK Signaling Pathways in Pancreatic Beta Cells: The Regulation of RAF Activation by Nutrient Stimuli(2011-02-01) Duan, Lingling; Cobb, Melanie H.In pancreatic β cells cells, ERK1 and ERK2 participate in nutrient sensing and their activities rise and fall as a function of glucose concentration over the physiologic range. Glucose metabolism triggers calcium influx and release of calcium from intracellular stores which are required for ERK1/2 activity. Calcium influx also activates the calcium-dependent phosphatase calcineurin, which is required for maximal ERK1/2 activation by glucose. Calcineurin controls insulin gene expression by ERK1/2-dependent and -independent mechanisms. This study showed that in β cells, glucose activates the ERK1/2 cascade primarily through B-Raf. Glucose also enhances dimerization of B-Raf with C-Raf. Furthermore, calcineurin up-regulates B-Raf activity and stabilizes C-Raf/B-Raf in response to glucose. Calcineurin binds to B-Raf in both unstimulated and stimulated cells. B-Raf phospho-T401 is one of the target sites that can be dephosphorylated by calcineurin. This study reveals that cross-talk between Raf and calcineurin is essential for the maximal activation of ERK1/2 in the glucose signaling pathways. [Keywords: TCF; POP-1; Wnt; embryogenesis; C. elegans]Item Physical and Functional Interaction Between Calcineurin and the Cardiac L-Type Calcium Channel(2008-09-19) Tandan, Samvit; Hill, Joseph A.The L-type Ca2+ channel is the major mediator of Ca2+ influx in cardiomyocytes leading to both mechanical contraction and activation of signaling cascades. Among these cascades is calcineurin, a protein phosphatase that promotes hypertrophic growth of the heart. We previously reported from in vivo models of pressure-overload that calcineurin regulates Ca2+ channel function in the heart, such that, inhibition of calcineurin activity results in a decrease in channel function. Based on this, we hypothesize that calcineurin participates in the channel macromolecular complex. Initial immunohistochemical data demonstrated evidence for co-localization of calcineurin to alpha1C, the pore-forming subunit of the L-type Ca2+ channel in mouse ventricular tissue. Additionally, co-immunoprecipitation biochemical experiments revealed evidence for binding between calcineurin and alpha1C in native mouse and rat heart tissues. Pulldown assays using GST-fusion proteins of all intracellular alpha1C regions provided evidence for direct binding of calcineurin at the N- and C-termini of alpha1C. At the C-terminus, calcineurin bound to aa 1909-2029 overlapping the well-characterized PKA/PKC site Ser-1928. In vitro kinase/phosphatase assays revealed Ser-1928 as a substrate for calcineurin dephosphorylation. Voltage-clamp recordings of L-type Ca2+ currents from cultured cardiomyocytes expressing constitutively-active calcineurin revealed significant up-regulation of channel function, similar to our previous observations from cardiac hypertrophy in vivo. Conversely, acute suppression of calcineurin, both pharmacologically or with specific peptide-inhibitors, induced a significant decrease in L-type channel function, while neither intervention had an effect on channel function in the absence of calcineurin activity. These data provide evidence for direct interaction between the L-type Ca2+ channel and calcineurin, and insights into the regulation of the channel by calcineurin. Furthermore, they highlight the specific role of calcineurin as a potential mediator of pathophysiological electrical remodeling in cardiac hypertrophy and failure.