Olga A. Akimova

Ubiquitous [Na+]i/[K+]i-Sensitive Transcriptome in Mammalian Cells: Evidence for Ca2+i-Independent Excitation-Transcription Coupling

Stimulus-dependent elevation of intracellular Ca2+ ([Ca2+]i) affects the expression of numerous genes – a phenomenon known as excitation-transcription coupling. Recently, we found that increases in [Na+]i trigger c-Fos expression via a novel Ca2+ i-independent pathway. In the present study, we identified ubiquitous and tissue-specific [Na+]i/[K+]i-sensitive transcriptomes by comparative analysis of differentially expressed genes in vascular smooth muscle cells from rat aorta (RVSMC), the human adenocarcinoma cell line HeLa, and human umbilical vein endothelial cells (HUVEC). To augment [Na+]i and reduce [K+]i, cells were treated for 3 hrs with the Na+,K+-ATPase inhibitor ouabain or placed for the same time in the K+-free medium. Employing Affymetrix-based technology, we detected changes in expression levels of 684, 737 and 1839 transcripts in HeLa, HUVEC and RVSMC, respectively, that were highly correlated between two treatments (p<0.0001; R2>0.62). Among these Na+ i/K+ i-sensitive genes, 80 transcripts were common for all three types of cells. To establish if changes in gene expression are dependent on increases in [Ca2+]i, we performed identical experiments in Ca2+-free media supplemented with extracellular and intracellular Ca2+ chelators. Surprisingly, this procedure elevated rather than decreased the number of ubiquitous and cell-type specific Na+ i/K+ i-sensitive genes. Among the ubiquitous Na+ i/K+ i-sensitive genes whose expression was regulated independently of the presence of Ca2+ chelators by more than 3-fold, we discovered several transcription factors (Fos, Jun, Hes1, Nfkbia), interleukin-6, protein phosphatase 1 regulatory subunit, dual specificity phosphatase (Dusp8), prostaglandin-endoperoxide synthase 2, cyclin L1, whereas expression of metallopeptidase Adamts1, adrenomedulin, Dups1, Dusp10 and Dusp16 was detected exclusively in Ca2+-depleted cells. Overall, our findings indicate that Ca2+ i-independent mechanisms of excitation-transcription coupling are involved in transcriptomic alterations triggered by elevation of the [Na+]i/[K+]i ratio. There results likely have profound implications for normal and pathological regulation of mammalian cells, including sustained excitation of neuronal cells, intensive exercise and ischemia-triggered disorders.

Transient activation and delayed inhibition of Na+,K+,Cl- cotransport in ATP-treated C11-MDCK cells involve distinct P2Y receptor subtypes and signaling mechanisms.

In C11-MDCK cells, which resemble intercalated cells from collecting ducts of the canine kidney, P2Y agonists promote transient activation of the Na+,K+,Cl– cotransporter (NKCC), followed by its sustained inhibition. We designed this study to identify P2Y receptor subtypes involved in dual regulation of this carrier. Real time polymerase chain reaction analysis demonstrated that C11-MDCK cells express abundant P2Y1 and P2Y2 mRNA compared with that of other P2Y receptor subtypes. The rank order of potency of agents (ATP ∼ UTP ≫ 2-(methylthio)-ATP (2MeSATP); adenosine 5′-[β-thio]diphosphate (ADPβS) inactive) indicated that P2Y2 rather than P2Y1 receptors mediate a 3–4-fold activation of NKCC within the first 5–10 min of nucleotide addition. NKCC activation in ATP-treated cells was abolished by the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, calmodulin (CaM) antagonists trifluoroperazine and W-7, and KN-62, an inhibitor of Ca2+/CaM-dependent protein kinase II. By contrast with the transient activation, 30-min incubation with nucleotides produced up to 4–5-fold inhibition of NKCC, and this inhibition exhibited a rank order of potency (2MeSATP > ADPβS > ATP ≫ UTP) typical of P2Y1 receptors. Unlike the early response, delayed inhibition of NKCC occurred in 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-loaded cells and was completely abolished by the P2Y1 antagonists MRS2179 and MRS2500. Transient activation and delayed inhibition of NKCC in C11 cell monolayers were observed after the addition of ATP to mucosal and serosal solutions, respectively. NKCC inhibition triggered by basolateral application of ADPβS was abolished by MRS2500. Our results thus show that transient activation and delayed inhibition of NKCC in ATP-treated C11-MDCK cells is mediated by Ca2+/CaM-dependent protein kinase II- and Ca2+-independent signaling triggered by apical P2Y2 and basolateral P2Y1 receptors, respectively.

Transcriptomic Changes Triggered by Hypoxia: Evidence for HIF-1α -Independent, [Na + ] i /[K + ] i -Mediated, Excitation-Transcription Coupling

This study examines the relative impact of canonical hypoxia-inducible factor-1alpha- (HIF-1α and Na+ i/K+ i-mediated signaling on transcriptomic changes evoked by hypoxia and glucose deprivation. Incubation of RASMC in ischemic conditions resulted in ∼3-fold elevation of [Na+]i and 2-fold reduction of [K+]i. Using global gene expression profiling we found that Na+,K+-ATPase inhibition by ouabain or K+-free medium in rat aortic vascular smooth muscle cells (RASMC) led to the differential expression of dozens of genes whose altered expression was previously detected in cells subjected to hypoxia and ischemia/reperfusion. For further investigations, we selected Cyp1a1, Fos, Atf3, Klf10, Ptgs2, Nr4a1, Per2 and Hes1, i.e. genes possessing the highest increments of expression under sustained Na+,K+-ATPase inhibition and whose implication in the pathogenesis of hypoxia was proved in previous studies. In ouabain-treated RASMC, low-Na+, high-K+ medium abolished amplification of the [Na+]i/[K+]i ratio as well as the increased expression of all tested genes. In cells subjected to hypoxia and glucose deprivation, dissipation of the transmembrane gradient of Na+ and K+ completely eliminated increment of Fos, Atf3, Ptgs2 and Per2 mRNAs and sharply diminished augmentation expression of Klf10, Edn1, Nr4a1 and Hes1. In contrast to low-Na+, high-K+ medium, RASMC transfection with Hif-1a siRNA attenuated increments of Vegfa, Edn1, Klf10 and Nr4a1 mRNAs triggered by hypoxia but did not impact Fos, Atf3, Ptgs2 and Per2 expression. Thus, our investigation demonstrates, for the first time, that Na+ i/K+ i-mediated, Hif-1α- -independent excitation-transcription coupling contributes to transcriptomic changes evoked in RASMC by hypoxia and glucose deprivation.

Cardiotonic Steroids: Novel Mechanisms of Na+ i-Mediated and - Independent Signaling Involved in the Regulation of Gene Expression, Proliferation and Cell Death

Vascular remodeling and hypertrophy/hyperplasia of the heart and kidney are known to be major servomechanisms of long-term maintenance of elevated blood pressure and the development of cardiovascular and renal complications seen in hypertension. Several lines of evidence suggest that these abnormalities are genetically determined and evoke an imbalance between cell proliferation and death. During the last 2 decades, it was shown that the level of cardiotonic steroids (CTS), i.e. potent and selective Na+,K+-ATPase inhibitors, is increased in several forms of volumeexpanded hypertension. We focus our review on recent data implicating CTS in the regulation of cell proliferation and death. At low concentrations, CTS augment proliferation of vascular smooth muscle cells (VSMC) as well as renal epithelial and vascular endothelial cells without significant inhibition of Na+,K+-ATPase-mediated ion fluxes. In contrast to cell proliferation, effect of CTS on cell survival is tissue-specific. In VSMC, CTS inhibit programmed cell death via a novel Na+ i-mediated, Ca2+ i-independent mechanism of expression of antiapoptotic genes including mortalin, whereas in vascular endothelial and renal epithelial cells, long-term exposure to CTS leads to cell death showing combined markers of apoptosis and necrosis. This mode of cell death is caused by interaction of CTS with Na+,K+-ATPase but is independent of inhibition of the Na+,K+-ATPase-mediated ion fluxes and inversion of the [Na+]i/[K+]i ratio. We propose that these novel signaling pathways triggered by enhanced production of endogenous CTS and/or abnormal Na+ handling contribute to cardiovascular and renal complications seen in hypertension.

Antifibrotic effects of noscapine through activation of prostaglandin E2 receptors and protein kinase A.

Background: Noscapine is a safe orally available anticough medicine also known to bind microtubules and induce cancer cell death. Results: Noscapine inhibits myofibroblast differentiation and pulmonary fibrosis through prostaglandin receptors and activation of PKA. Conclusion: Noscapine is an antifibrotic drug acting through PKA activation via EP2 prostaglandin receptors. Significance: This study describes a novel antifibrotic function and novel mechanism of action of noscapine. Myofibroblast differentiation is a key process in the pathogenesis of fibrotic disease. We have shown previously that differentiation of myofibroblasts is regulated by microtubule polymerization state. In this work, we examined the potential antifibrotic effects of the antitussive drug, noscapine, recently found to bind microtubules and affect microtubule dynamics. Noscapine inhibited TGF-β-induced differentiation of cultured human lung fibroblasts (HLFs). Therapeutic noscapine treatment resulted in a significant attenuation of pulmonary fibrosis in the bleomycin model of the disease. Noscapine did not affect gross microtubule content in HLFs, but inhibited TGF-β-induced stress fiber formation and activation of serum response factor without affecting Smad signaling. Furthermore, noscapine stimulated a rapid and profound activation of protein kinase A (PKA), which mediated the antifibrotic effect of noscapine in HLFs, as assessed with the PKA inhibitor, PKI. In contrast, noscapine did not activate PKA in human bronchial or alveolar epithelial cells. Finally, activation of PKA and the antifibrotic effect of noscapine in HLFs were blocked by the EP2 prostaglandin E2 receptor antagonist, PF-04418948, but not by the antagonists of EP4, prostaglandin D2, or prostacyclin receptors. Together, we demonstrate for the first time the antifibrotic effect of noscapine in vitro and in vivo, and we describe a novel mechanism of noscapine action through EP2 prostaglandin E2 receptor-mediated activation of PKA in pulmonary fibroblasts.

The death of ouabain-treated renal epithelial cells: evidence against anoikis occurrence

AbstractThe mechanisms of cell death signaling triggered by cardiotonic steroids are poorly understood. Based on massive detachment of ouabain-treated Madin-Darby canine kidney (MDCK) cells, it may be proposed that the cytotoxic action of these compounds is mediated by anoikis, i.e. a particular mode of death occurring in cells lacking cell-to-extracellular matrix interactions. We tested this hypothesis. Six hour incubation of MDCK cells with ouabain, marinobufagenin or K+-free medium almost completely blocked Na+,K+-ATPase, increased Nai+ content by ∼10-fold and suppressed cell attachment to regular-plastic-plates by up to 5-fold. In contrast, the death of attached cells was observed after 24-h incubation with ouabain but not in the presence of marinobufagenin or K+-free medium. Cells treated with ouabain and undergoing anoikis on ultra-low attachment plates exhibited different cell volume behaviour, i.e. swelling and shrinkage, respectively. The pan-caspase inhibitor z-VAD.fmk and the protein kinase C activator PMA rescued MDCK cells from anoikis but did not influence the survival of ouabain-treated cells, whereas medium acidification from pH 7.2 to 6.7 almost completely abolished the cytotoxic action of ouabain, but did not significantly affect anoikis. Our results show that the Na i+,Ki+-independent mode of MDCK cell death evoked by ouabain is not mediated by anoikis.

[Na+]i‐induced c‐Fos expression is not mediated by activation of the 5′‐promoter containing known transcriptional elements

In vascular smooth muscle cells and several other cell types, inhibition of Na+/K+‐ATPase leads to the expression of early response genes, including c‐Fos. We designed this study to examine whether or not a putative Na+i/K+i‐sensitive element is located within the c‐Fos 5′‐UTR from − 650 to + 103 containing all known response elements activated by ‘classic’ stimuli, such as growth factors and Ca2+i‐raising compounds. In HeLa cells, the highest increment of c‐Fos mRNA content was noted after 6 h of Na+/K+‐ATPase inhibition with ouabain that was abolished by actinomycin D, an inhibitor of RNA synthesis. c‐Fos protein accumulation in ouabain‐treated cells correlated with a gain of Na+i and loss of K+i. Augmented c‐Fos expression was also observed under inhibition of Na+/K+‐ATPase in K+‐free medium and in the presence of the Na+ ionophore monensin. The effect of ouabain on c‐Fos expression was sharply attenuated under dissipation of the transmembrane Na+ gradient, but was preserved in the presence of Ca2+ chelators and the extracellular regulated kinase inhibitor PD98059, thus indicating an Na+i‐mediated, Ca2+i‐ and extracellular regulated kinase‐independent mechanism of gene expression. In contrast to massive c‐Fos expression, we failed to detect any effect of ouabain on accumulation of luciferase driven by the c‐Fos 5′‐UTR. Negative results were also obtained in ouabain‐treated vascular smooth muscle cells and C11 Madin–Darby canine kidney cells possessing augmented c‐Fos expression. Our results reveal that Na+i‐induced c‐Fos expression is not mediated by the 5′‐UTR containing transcriptional elements activated by growth factors and other ‘classic stimuli’.

Regulation of myofibroblast differentiation by cardiac glycosides

Myofibroblast differentiation is a key process in pathogenesis of fibrotic diseases. Cardiac glycosides (ouabain, digoxin) inhibit Na(+)-K(+)-ATPase, resulting in increased intracellular [Na(+)]-to-[K(+)] ratio in cells. Microarray analysis suggested that increased intracellular [Na(+)]/[K(+)] ratio may promote the expression of cyclooxygenase-2 (COX-2), a critical enzyme in the synthesis of prostaglandins. Given antifibrotic effects of prostaglandins through activation of protein kinase A (PKA), we examined if cardiac glycosides stimulate COX-2 expression in human lung fibroblasts and how they affect myofibroblast differentiation. Ouabain stimulated a profound COX-2 expression and a sustained PKA activation, which was blocked by COX-2 inhibitor or by COX-2 knockdown. Ouabain-induced COX-2 expression and PKA activation were abolished by the inhibitor of the Na(+)/Ca(2+) exchanger, KB-R4943. Ouabain inhibited transforming growth factor-β (TGF-β)-induced Rho activation, stress fiber formation, serum response factor activation, and the expression of smooth muscle α-actin, collagen-1, and fibronectin. These effects were recapitulated by an increase in intracellular [Na(+)]/[K(+)] ratio through the treatment of cells with K(+)-free medium or with digoxin. Although inhibition of COX-2 or of the Na(+)/Ca(2+) exchanger blocked ouabain-induced PKA activation, this failed to reverse the inhibition of TGF-β-induced Rho activation or myofibroblast differentiation by ouabain. Together, these data demonstrate that ouabain, through the increase in intracellular [Na(+)]/[K(+)] ratio, drives the induction of COX-2 expression and PKA activation, which is accompanied by a decreased Rho activation and myofibroblast differentiation in response to TGF-β. However, COX-2 expression and PKA activation are not sufficient for inhibition of the fibrotic effects of TGF-β by ouabain, suggesting that additional mechanisms must exist.

Decreased NKCC1 Activity in Erythrocytes From African Americans With Hypertension and Dyslipidemia

BACKGROUND Recent studies demonstrated a key role of ubiquitous isoform of Na+,K+,2Cl- co-transport (NKCC1) in regulation of myogenic tone and peripheral resistance. We examined the impact of race, gender, and plasma lipid on NKCC1 activity in French Canadians and African Americans with hypertension and dyslipidemia. METHODS NKCC and passive erythrocyte membrane permeability to K+, measured as ouabain-resistant, bumetanide-sensitive, and (ouabain+bumetanide)-resistant 86Rb influx, respectively, were compared in 111 French-Canadian men, 107 French-Canadian women, 26 African-American men, and 45 African-American women with essential hypertension and dyslipidemia. RESULTS The African-American men and women were 7 years younger and presented twofold decreased plasma triglycerides compared to their French-Canadian counterparts (P < 0.01) whereas body mass index (BMI), total cholesterol, low-density lipoprotein, and high-density lipoprotein (HDL) were not different. NKCC was respectively 50 and 38% lower in the African-American men and women than in the French Canadians (P < 0.005) without any differences in passive erythrocyte membrane permeability for K+. We did not observe any impact of age on NKCC in all groups under investigation, whereas plasma triglycerides correlated positively with the activity of this carrier in the French-Canadian men only. CONCLUSIONS NKCC1 activity is lower in erythrocytes of African Americans with essential hypertension and dyslipidemia than in Caucasian counterparts. We suggest that decreased NKCC1 may contribute to the feature of the pathogenesis of salt-sensitive hypertension seen in African Americans.

Purinergic inhibition of Na(+),K (+),Cl (-) cotransport in C11-MDCK cells: Role of stress-activated protein kinases

Previously, we observed that sustained activation of P2Y1 leads to inhibition of Na+,K+,Cl− cotransport (NKCC) in C11 cells resembling intercalated cells from collecting ducts of the Madin-Darby canine kidney. This study examined the role of stress-activated protein kinases (SAPK) in NKCC inhibition triggered by purinergic receptors. Treatment of C11 cells with ATP led to sustained phosphorylation of SAPK such as JNK and p38. Activation of these kinases also occurred in anisomycin-treated cells. Surprisingly, we observed that compounds SP600125 and SB202190, known as potent inhibitors of JNK and p38 in cell-free systems, activated rather than inhibited phosphorylation of the kinases in C11 cells. Importantly, similarly to ATP, all the above-listed activators of JNK and p38 phosphorylation inhibited NKCC. Thus, our results suggest that activation of JNK and/or p38 contributes to NKCC suppression detected in intercalated-like cells from distal tubules after their exposure to P2Y1 agonists.