Identification of ryanodine receptor isoforms in prostate DU-145, LNCaP, and PWR-1E cells

We previously reported that left ventricular (LV) slices from isoproterenol (ISO)-induced hypertrophied rat hearts showed an increase of energy expenditure due to remodeling of Ca(2+) handling in excitation-contraction coupling, i.e., suppressed SERCA2a activity and enhanced Na(+)/Ca(2+)exchanger-1 (NCX-1) activity. Na(+)/H(+) exchanger-1 (NHE-1) inhibitor (NHEI) has been demonstrated to exert beneficial effects in the development of cardiac remodeling. We hypothesized that a novel NHE-1 selective inhibitor, BIIB723 prevents remodeling of Ca(2+) handling in LV slices of ISO-induced hypertrophied rat hearts mediated by inhibiting NCX-1 activity. The significant shortening in duration of multi-cellular Ca(2+) transient in ISO group was normalized in ISO+BIIB723 group. The significant increase in amplitude of multi-cellular Ca(2+) waves (CaW) generated at high [Ca(2+)](o) of LV slices in ISO group was also normalized in ISO+BIIB723 group. However, the enhanced NCX-1 activity was not antagonized by BIIB723. We recently reported that ISO-induced down-regulation of a Ca(2+) handling protein, SERCA2a, was normalized by BIIB723. Therefore, it seems likely that BIIB723 normalized shortened multi-cellular Ca(2+) transient duration and increased CaW amplitude in LV slices mediated via normalization of SERCA2a activity. Furthermore, the results presented here suggest the multi-cellular Ca(2+) transient duration and CaW amplitude in LV slices might be better indices reflecting SERCA2a activity than SERCA2a protein expression level.     

Calcium sensing receptor regulates cardiomyocyte function through nuclear calcium

Nuclear Ca2+ plays a pivotal role in the regulation of gene expression. IP3 (inositol-1,4,5-trisphosphate) is an important regulator of nuclear Ca2+. We hypothesized that the CaR (calcium sensing receptor) stimulates nuclear Ca2+ release through IICR (IP3-induced calcium release) from perinuclear stores. Spontaneous Ca2+ oscillations and the spark frequency of nuclear Ca2+ were measured simultaneously in NRVMs (neonatal rat ventricular myocytes) using confocal imaging. CaR-induced nuclear Ca2+ release through IICR was abolished by inhibition of CaR and IP3Rs (IP3 receptors). However, no effect on the inhibition of RyRs (ryanodine receptors) was detected. The results suggest that CaR specifically modulates nuclear Ca2+ signalling through the IP3R pathway. Interestingly, nuclear Ca2+ was released from perinuclear stores by CaR activator-induced cardiomyocyte hypertrophy through the Ca2+-dependent phosphatase CaN (calcineurin)/NFAT (nuclear factor of activated T-cells) pathway. We have also demonstrated that the activation of the CaR increased the NRVM protein content, enlarged cell size and stimulated CaN expression and NFAT nuclear translocation in NRVMs. Thus, CaR enhances the nuclear Ca2+ transient in NRVMs by increasing fractional Ca2+ release from perinuclear stores, which is involved in cardiac hypertrophy through the CaN/NFAT pathway.     

NHE-1 participates in isoproterenol-induced downregulation of SERCA2a and development of cardiac remodeling in rat hearts

Impaired Ca(2+) handling is one of the main characteristics in heart failure patients. Recently, we reported abnormal expressions of Ca(2+)-handling proteins in isoproterenol (ISO)-induced hypertrophied rat hearts. On the other hand, Na(+)/H(+) exchanger (NHE)-1 inhibitor has been demonstrated to exert beneficial effects in ischemic-reperfusion injury and in the development of cardiac remodeling. The aims of the present study are to investigate the role of NHE-1 on Ca(2+) handling and development of cardiac hypertrophy in ISO-infused rats. Male Wistar rats were randomly divided into vehicle [control (CTL)] and ISO groups without or with pretreatment with a selective NHE-1 inhibitor, BIIB-723. ISO infusion for 1 wk significantly increased the ratios of heart to body weight and left ventricle (LV) to body weight and collagen accumulation. All of these increases were antagonized by coadministration with BIIB-723. The ISO-induced significant increase in LV wall thickness was suppressed significantly (P < 0.05) by BIIB-723. ISO-induced decreases in cardiac stroke volume and a total mechanical energy per beat index, systolic pressure-volume area at midrange LV volume, were normalized by BIIB-723. The markedly higher expression of NHE-1 protein in the ISO group than that in CTL group was suppressed (P < 0.05) by BIIB-723. Surprisingly, ISO induced downregulation of the important Ca(2+)-handling protein sarcoplasmic reticulum Ca(2+)-ATPase 2a, the expression of which was also normalized by BIIB-723 without changes in phosphorylated phospholamban (PLB)/PLB expression. We conclude that NHE-1 contributes to ISO-induced abnormal Ca(2+) handling associated with cardiac hypertrophy. Inhibition of NHE-1 ameliorates cardiac Ca(2+)-handling impairment and prevents the development of cardiac dysfunction in ISO-infused rats.     

钙调神经磷酸酶信号通路相关抑制因子研究进展

钙调神经磷酸酶(calcineurin,CaN)信号通路是介导心肌肥厚的一条重要通路,随着研究的不断深入, 其相关抑制因子的研究也受到了更多关注.综述了Ca2+CaN-NFAT信号通路上、下游及CaN本身的部分抑制因子在抗心肌肥大过程中的作用,这些因子的研究开发对心肌肥厚的治疗具有重要意义.     

钠氢交换体1在病理性心肌肥厚大鼠心肌组织及血浆中的表达

目的：研究异丙肾上腺素诱导的病理性心肌肥厚大鼠心肌组织及血浆中钠氢交换体1（sodium—hydrogen exchanger1,NHE—1）的表达,探讨NHE1在心肌肥厚发生和发展中的作用。方法：30只雄性SD大鼠随机并平均分为2组：病理性心肌肥厚组和对照组,每组15只,病理性心肌肥厚组（以下简称ISO组）予以ISO（异丙肾上腺素）连续每日以20、10和5mg／kg的剂量递减皮下注射,再以3mg／kg的剂量维持皮下注射7d,对照组予相同剂量生理盐水皮下注射。给药结束后进行心脏超声检测左室舒张末径（LVEDD）、左室收缩末径（LVESD）、室间隔厚度（IVST）、短轴缩短率（FS）、左室射血分数（LVEF）。分别测定各组大鼠体重（Bw）、心室重量（VW）、左心室重量（LVW）,计算心室重量指数VWI（VW／BW）、左心室重量指数LVWI（LVW／BW）。取血检测血浆中NHE．1的浓度,并取心肌组织观察病理形态学特征,用免疫组化法检测心肌组织中NHE—1的表达量。结果：与对照组相比,ISO组大鼠LVEF、IVST显著增加（P〈0．05）,LVESD明显降低（P〈0．05）,VWI、LVWI明显增加（P〈0．01）,血浆NHE—1浓度明显升高（P〈0．01）,心肌组织NHE-1表达增多（P〈0．01）。结论：NHE-1可能在病理性心肌肥厚的发生和发展过程中起着重要作用。     

Caveolin-3 Overexpression Attenuates Cardiac Hypertrophy via Inhibition of T-type Ca2+ Current Modulated by Protein Kinase Cα in Cardiomyocytes

Pathological cardiac hypertrophy is characterized by subcellular remodeling of the ventricular myocyte with a reduction in the scaffolding protein caveolin-3 (Cav-3), altered Ca²⁺ cycling, increased protein kinase C expression, and hyperactivation of calcineurin/nuclear factor of activated T cell (NFAT) signaling. However, the precise role of Cav-3 in the regulation of local Ca²⁺ signaling in pathological cardiac hypertrophy is unclear. We used cardiac-specific Cav-3-overexpressing mice and in vivo and in vitro cardiac hypertrophy models to determine the essential requirement for Cav-3 expression in protection against pharmacologically and pressure overload-induced cardiac hypertrophy. Transverse aortic constriction and angiotensin-II (Ang-II) infusion in wild type (WT) mice resulted in cardiac hypertrophy characterized by significant reduction in fractional shortening, ejection fraction, and a reduced expression of Cav-3. In addition, association of PKCα and angiotensin-II receptor, type 1, with Cav-3 was disrupted in the hypertrophic ventricular myocytes. Whole cell patch clamp analysis demonstrated increased expression of T-type Ca²⁺ current (I_{Ca, T}) in hypertrophic ventricular myocytes. In contrast, the Cav-3-overexpressing mice demonstrated protection from transverse aortic constriction or Ang-II-induced pathological hypertrophy with inhibition of I_{Ca, T} and intact Cav-3-associated macromolecular signaling complexes. siRNA-mediated knockdown of Cav-3 in the neonatal cardiomyocytes resulted in enhanced Ang-II stimulation of I_{Ca, T} mediated by PKCα, which caused nuclear translocation of NFAT. Overexpression of Cav-3 in neonatal myocytes prevented a PKCα-mediated increase in I_{Ca, T} and nuclear translocation of NFAT. In conclusion, we show that stable Cav-3 expression is essential for protecting the signaling mechanisms in pharmacologically and pressure overload-induced cardiac hypertrophy.     

Na+/H+ exchange inhibitors: a new class of cardioprotectors

The Na+/H+ exchanger (NHE) extrudes intracellular H+ in exchange for Na+ in an electroneutral process. Of the 6 mammalian exchanger isoforms identified to date, the NHE-1 is believed to be the molecular homologue of the sarcolemma Na+/H+ transporter. The exchanger is activated primarily by a reduction in intracellular pH, although such activation is subject to modulation by a variety of endogenous mediators (catecholamines, thrombin, endothelin) through receptor-mediated mechanisms. A large body of animal studies using both in vitro and in vivo models indicates that the inhibition of the sarcolemma NHE-1 attenuates myocardial injury in ischemia and reperfusion. Cardioprotective effects of NHE-1 inhibition involve a reduced susceptibility to severe ventricular arrhythmia, augmentation of contractile function recovery, and limitation of infarction size during reperfusion. Such protection is likely to arise partly from attenuation of "Ca2+ overload" in ischemic cardiomyocytes, which has been causally linked with all these pathologic phenomena. A marked benefit that has been observed with cariporide (HOE-642) and its structurally related congener HOE-694 in patients with acute myocardial infarction and in cardiac surgery demonstrates that selective NHE-1 inhibitors represent a novel and effective class of cardioprotectors.     

MLP参与心肌肥大发生过程与Calcineurin/NFAT信号通路有关

心肌肥大是心肌细胞面对多种病理刺激时的共同反应,以心肌细胞体积增大和胚胎期基因的重新表达为标志.心肌发育调控基因肌肉LIM蛋白(muscle LIM protein,MLP)的表达异常与心肌肥大有关.为研究MLP参与心肌肥大发生的分子机制,采用去氧肾上腺素（phenylephrine, PE）刺激大鼠原代培养心肌细胞,建立心肌细胞肥大模型,采用RNAi技术敲减MLP的表达,分析MLP与肥大信号通路钙调神经磷酸酶（calcineurin）/活化T细胞核因子（nuclear factor of activated T-cells, NFAT）的关系.结果显示, 原代培养的心肌细胞经一定浓度的PE刺激后细胞表面积增加,肥大标志蛋白ANP、BNP表达增高,并伴有MLP表达上调. RNAi方法敲减MLP的表达则明显抑制PE诱导的心肌细胞表面积增加和BNP表达增高,并且直接 影响NFAT的转录激活活性,提示MLP与心肌肥大的发生密切相关,并且可能是通过calcineurin/NFAT信号通路而参与心肌肥大的发生.     

Activation of peroxisome proliferator-activated receptor gamma inhibits endothelin-1-induced cardiac hypertrophy via the calcineurin/NFAT signaling pathway

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) has been described as a negative regulator of cardiac hypertrophy. A better understanding of PPAR-gamma and cardiac hypertrophy may facilitate the development of novel therapeutic strategies to treat heart diseases related to cardiac hypertrophy by mimicking the naturally preferred mechanisms. In the present study, we investigated the interaction between PPAR-gamma and calcineurin/nuclear factor of activated T-cells (NFAT) in endothelin-1 (ET-1)-induced hypertrophy of neonatal rat cardiac myocytes. The results suggest that the treatment of cultured cardiac myocytes with a PPAR-gamma ligand, rosiglitazone, inhibited the ET-1-induced increase in protein synthesis, surface area, calcineurin enzymatic activity, and protein expression. Both the application of rosiglitazone and overexpression of the PPAR-gamma inhibited the nuclear translocation of NFATc4. Moreover, co-immunoprecipitation studies showed that rosiglitazone enhanced the association between PPAR-gamma and calcineurin/NFAT. These results suggest that ET-1-induced cardiac hypertrophy is inhibited by activation of PPAR-gamma, which is at least partly due to cross-talk between PPAR-gamma and calcineurin/NFAT.     

Acetaldehyde depresses myocardial contraction and cardiac myocyte shortening in spontaneously hypertensive rats: role of intracellular Ca2+.

Acetaldehyde (ACA), the major metabolite of ethanol, exerts both stimulatory and depressive actions on myocardial tissue. We have recently shown that ACA depresses myocardial contraction, cardiac myocyte shortening and intracellular Ca2+ transients in normal rat heart. The purpose of the present study was to determine the influence of hypertension on ACA-induced myocardial actions. Mechanical properties of left ventricular papillary muscles and ventricular myocytes isolated from both 25-week-old normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) were evaluated using force-transducer and video edge-detection, respectively. Papillary muscles and cardiac myocytes were electrically stimulated to contract at 0.5 Hz. Contractile properties analyzed include: peak tension development (PTD), peak twitch amplitude (PTA), time-to-PTD/PTA (TPT/TPS), time-to-90% relaxation/relengthening (RT90/TR90) and maximal velocities of contraction/shortening and relaxation/relengthening (+/-VT/+/-dL/dt). Intracellular Ca2+ transients were measured as fura-2 fluorescence intensity (FFI) changes. ACA (1-30 mM) depressed PTD without affecting other mechanical indices in both WKY and SHR myocardium, with maximal inhibition of 64 and 69%, respectively. SHR myocytes exhibited increased cell dimension, baseline PTA and resting intracellular Ca2+ levels, compared to WKY counterparts. ACA (0.03-30 mM) depressed PTA without affecting TPT, TR90 and +/-dL/dt. The maximal inhibitions were 31 and 36% in WKY and SHR groups, respectively. Interestingly, ACA exerted a biphasic effect on FFI, displaying potentiation at lower doses (<3 mM) and inhibition at higher doses (>3 mM). The maximal increase in FFI changes were 19 and 22% at 0.3 mM and the maximal decreases were 37 and 29% at 30 mM ACA, in WKY and SHR myocytes, respectively. Neither resting intracellular Ca2+ levels (FFI) nor fluorescence decay time (FDT) were affected by ACA. The increase in FFI was attenuated by propranolol (1 microM), whereas the decrease in FFI was reversed by BayK 8644 (1 microM). These results suggest that hypertension does not appear to alter ACA-induced myocardial depression. The mechanism underlying ACA-induced myocardial actions may involve increased beta-adrenergic activity at low doses and reduced Ca2+ entry and/or release at high doses.     

Involvement of the Na+-independent Cl-/HCO3- exchange (AE) isoform in the compensation of myocardial Na+/H+ isoform 1 hyperactivity in spontaneously hypertensive rats

Enhanced activity of Na+/H+ isoform 1 (NHE-1) and the Na+-independent Cl-/HCO3- exchange (AE) is a feature of the hypertrophied myocardium in spontaneously hypertensive rats (SHR). The present study explored the possibility that sustained intracellular acidosis due to increased myocardial acid loading through AE causes NHE-1 enhancement. To this aim, SHR were treated for 2 weeks with a rabbit polyclonal antibody against an AE3 isoform that was recently developed and proven to have inhibitory effects on myocardial AE activity. We then compared the AE activity in the left ventricle papillary muscles isolated from untreated SHR with antiAE3-treated SHR; AE activity was measured in terms of the rate of intracellular pH recovery after an intracellular alkali load was introduced. AE activity was diminished by approximately 70% in SHR treated with the antiAE3 antibody, suggesting that the AE3 isoform is a major carrier of acid-equivalent influx in the hypertrophied myocardium. However, the antibody treatment failed to normalize NHE-1 activity that remained elevated in the myocardium of normotensive rats. The data therefore rule out the possibility that NHE-1 hyperactivity in hypertensive myocardium was due to sustained intracellular acidosis induced by increased AE activity that characterizes SHR myocardial tissue.     

钠氢交换体1 在病理性心肌肥厚大鼠心肌组织及血浆中的表达

目的:研究异丙肾上腺素诱导的病理性心肌肥厚大鼠心肌组织及血浆中钠氢交换体1(sodium-hydrogen exchanger 1,NHE-1)的表达,探讨NHE1在心肌肥厚发生和发展中的作用。方法:30只雄性SD大鼠随机并平均分为2组:病理性心肌肥厚组和对照组,每组15只,病理性心肌肥厚组(以下简称ISO组)予以ISO(异丙肾上腺素)连续每日以20、10和5mg/kg的剂量递减皮下注射,再以3mg/kg的剂量维持皮下注射7d,对照组予相同剂量生理盐水皮下注射。给药结束后进行心脏超声检测左室舒张末径(LVEDD)、左室收缩末径(LVESD)、室间隔厚度(IVST)、短轴缩短率(FS)、左室射血分数(LVEF)。分别测定各组大鼠体重(BW)、心室重量(VW)、左心室重量(LVW),计算心室重量指数VWI(VW/BW)、左心室重量指数LVW(ILVW/BW)。取血检测血浆中NHE-1的浓度,并取心肌组织观察病理形态学特征,用免疫组化法检测心肌组织中NHE-1的表达量。结果:与对照组相比,ISO组大鼠LVEF、IVST显著增加(P<0.05),LVESD明显降低(P<0.05),VWI、LVWI明显增加(P<0.01),血浆NHE-1浓度明显升高(P<0.01),心肌组织NHE-1表达增多(P<0.01)。结论:NHE-1可能在病理性心肌肥厚的发生和发展过程中起着重要作用。     

Decreased Ca2+ extrusion via Na+/Ca2+ exchange in epicardial left ventricular myocytes during compensated hypertrophy

Hypertension-induced cardiac hypertrophy alters the amplitude and time course of the systolic Ca2+ transient of subepicardial and subendocardial ventricular myocytes. The present study was designed to elucidate the mechanisms underlying these changes. Myocytes were isolated from the left ventricular subepicardium and subendocardium of 20-wk-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto rats (WKY; control). We monitored intracellular Ca2+ using fluo 3 or fura 2; caffeine (20 mmol/l) was used to release Ca2+ from the sarcoplasmic reticulum (SR), and Ni2+ (10 mM) was used to inhibit Na+/Ca2+ exchange (NCX) function. SHR myocytes were significantly larger than those from WKY hearts, consistent with cellular hypertrophy. Subepicardial myocytes from SHR hearts showed larger Ca2+ transient amplitude and SR Ca2+ content and less Ca2+ extrusion via NCX compared with subepicardial WKY myocytes. These parameters did not change in subendocardial myocytes. The time course of decline of the Ca2+ transient was the same in all groups of cells, but its time to peak was shorter in subepicardial cells than in subendocardial cells in WKY and SHR and was slightly prolonged in subendocardial SHR cells compared with WKY subendocardial myocytes. It is concluded that the major change in Ca2+ cycling during compensated hypertrophy in SHR is a decrease in NCX activity in subepicardial cells; this increases SR Ca2+ content and hence Ca2+ transient amplitude, thus helping to maintain the strength of contraction in the face of an increased afterload.     

蛋白激酶C对自发性高血压大鼠肥大心肌细胞无负荷收缩功能的调节

在慢性压力超负荷引起心肌肥大过程中,蛋白激酶C(protein kinase C,PKC)的激活起关键性作用,激活的PKC也能调节心肌收缩性能.本文旨在研究自发性高血压大(spontaneously hypertensive rat,SHR)心肌肥大的不同阶段PKC调节心肌收缩性能的特征.采用胶原酶法分离4月龄与10月龄Wistar-Kyoto(WKY)、SHR大鼠的心肌细胞,观测单个心肌细胞无负荷缩短幅值以及在PKC激动剂与抑制剂作用下心肌收缩性能的变化.结果表明:刺激频率从1 Hz增至3 Hz,WKY大鼠心肌细胞无负荷缩短幅值逐渐增加,呈正阶梯效应;4月龄SHR大鼠心肌细胞的缩短幅值较WKY大鼠增强,但在各刺激频率下其缩短幅值基本保持不变;10月龄SHR大鼠心肌细胞的缩短幅值在1 Hz刺激条件下与WKY大鼠无差别,随刺激频率增加,缩短幅值降低,呈负阶梯效应.在PKC激动剂PMA灌流条件下,50、100与200 nmol/L的PMA分别降低WKY大鼠心肌细胞缩短幅值至(69.8±1.9)%、(58.2 2.2)%与(22.7±2.5)%(均P<0.01),呈浓度依赖关系;PMA对4月龄SHR大鼠心肌细胞缩短幅值的降低更明显,分别降至(6.1±0.7)%、(2.4±0.2)%与(12.5±2.6)%(均P<0.01);PMA降低10月龄SHR大鼠心肌细胞缩短幅值至(65.7±1.6)%、(53.9±4.0)%与(16.3±2.0)%(均P<0.01),小于对4月龄SHR大鼠心肌细胞缩短幅值的作用.PKC抑制剂staurosporine增加WKY大鼠心肌细胞缩短幅值,在200 nmol/L的staurosporine灌流条件下,WKY大鼠、4月龄SHR大鼠、10月龄SHR大鼠心肌细胞缩短幅值分别增JJH(63.63±4.53)%、(80.82±4.61)%、(80.97±4.59)%(均P<0.05).结果提示,在SHR大鼠心肌肥大初期,具有负性肌力作用的PKC异构体可能被激活,并参与对心肌收缩性能的调节;而心肌肥大稳定阶段,这些PKC活性可能恢复至正常水平.     

Overexpression of type 1 angiotensin II receptors impairs excitation-contraction coupling in the mouse heart

Transgenic mice that overexpress human type 1 angiotensin II receptor (AT(1)R) in the heart develop cardiac hypertrophy. Previously, we have shown that in 6-mo AT(1)R mice, which exhibit significant cardiac remodeling, fractional shortening is decreased. However, it is not clear whether altered contractility is attributable to AT(1)R overexpression or is secondary to cardiac hypertrophy/remodeling. Thus the present study characterized the effects of AT(1)R overexpression on ventricular L-type Ca(2+) currents (I(CaL)), cell shortening, and Ca(2+) handling in 50-day and 6-mo-old male AT(1)R mice. Echocardiography showed there was no evidence of cardiac hypertrophy in 50-day AT(1)R mice but that fractional shortening was decreased. Cellular experiments showed that cell shortening, I(CaL), and Ca(v)1.2 mRNA expression were significantly reduced in 50-day and 6-mo-old AT(1)R mice compared with controls. In addition, Ca(2+) transients and caffeine-induced Ca(2+) transients were reduced whereas the time to 90% Ca(2+) transient decay was prolonged in both age groups of AT(1)R mice. Western blot analysis revealed that sarcoplasmic reticulum Ca(2+)-ATPase and Na(+)/Ca(2+) exchanger protein expression was significantly decreased in 50-day and 6-mo AT(1)R mice. Overall, the data show that cardiac contractility and the mechanisms that underlie excitation-contraction coupling are altered in AT(1)R mice. Furthermore, since the alterations in contractility occur before the development of cardiac hypertrophy, it is likely that these changes are attributable to the increased activity of the renin-angiotensin system brought about by AT(1)R overexpression. Thus it is possible that AT(1)R blockade may help maintain cardiac contractility in individuals with heart disease.     

Protein kinase-mediated regulation of calcineurin through the phosphorylation of modulatory calcineurin-interacting protein 1

Calcineurin is a serine/threonine protein phosphatase that plays a critical role in many physiologic processes such as T-cell activation, skeletal myocyte differentiation, and cardiac hypertrophy. We previously showed that active MEKK3 is capable of stimulating calcineurin/nuclear factor of activated T-cells (NFAT) signaling in cardiac myocytes through phosphorylation of modulatory calcineurin-interacting protein 1 (MCIP1). However, the protein kinases that function downstream of MEKK3 to mediate MCIP1 phosphorylation and the mechanism of MCIP1-mediated calcineurin regulation have not been defined. Here, we show that MEK5 and big MAP kinase 1 (BMK1) function downstream of MEKK3 in a signaling cascade that induces calcineurin activity through phosphorylation of MCIP1. Genetic studies showed that BMK1-deficient mouse lung fibroblasts failed to mediate MCIP1 phosphorylation and activate calcineurin/NFAT in response to angiotensin II, a potent NFAT activator. Conversely, restoring BMK1 to the deficient cells restored angiotensin II-mediated calcineurin/NFAT activation. Thus, using BMK1-deficient mouse lung fibroblast cells, we provided the genetic evidence that BMK1 is required for angiotensin II-mediated calcineurin/NFAT activation through MICP1 phosphorylation. Finally, we discovered that phosphorylated MCIP1 dissociates from calcineurin and binds with 14-3-3, thereby relieving its inhibitory effect on calcineurin activity. In summary, our findings reveal a previously unrecognized essential regulatory role of mitogen-activated protein kinase signaling in calcineurin activation through the reversible phosphorylation of a calcineurin-interacting protein, MCIP1.