In pressure or volume overload, hypertrophic growth of the myocardium is associated with myofibroblast differentiation, a process in which cardiac fibroblasts express smooth muscle α-actin (SMA). The signaling mechanisms that mediate force-induced myofibroblast differentiation and SMA expression are not defined. We examined the role of the Rho–Rho-kinase pathway in force-induced SMA expression in fibroblasts using an in vitro model system that applies static tensile forces (0.65 pN/μm2) to integrins via collagen-coated magnetite beads. Force maximally induced RhoA activation at 10 minutes that was localized to force application sites and required intact actin filaments. Force application induced phosphorylation of LIM kinase (5-10 minutes) and an early dephosphorylation of cofilin (5 minutes) that was followed by prolonged cofilin phosphorylation. These responses were blocked by Y27632, an inhibitor of Rho kinase. Force promoted actin filament assembly at force application sites (10-20 minutes), a process that required Rho kinase and cofilin. Force application induced nuclear translocation of the transcriptional co-activator MRTF-A but not MRTF-B. Nuclear translocation of MRTF-A required Rho kinase and intact actin filaments. Force caused 3.5-fold increases of SMA promoter activity that were completely blocked by transfection of cells with dominant-negative MRTF-A or by inhibition of Rho kinase or by actin filament disassembly. These data indicate that mechanical forces mediate actin assembly through the Rho–Rho-kinase–LIMK cofilin pathway. Force-mediated actin filament assembly promotes nuclear translocation of MRTF and subsequent activation of the SMA promoter to enhance SMA expression.

Osmotic stress is known to affect the cytoskeleton; however, this adaptive response has remained poorly characterized, and the underlying signaling pathways are unexplored. Here we show that hypertonicity induces submembranous de novo F-actin assembly concomitant with the peripheral translocation and colocalization of cortactin and the actin-related protein 2/3 (Arp2/3) complex, which are key components of the actin nucleation machinery. Additionally, hyperosmolarity promotes the association of cortactin with Arp2/3 as revealed by coimmunoprecipitation. Using various truncation or phosphorylation-incompetent mutants, we show that cortactin translocation requires the Arp2/3- or the F-actin binding domain, but the process is independent of the shrinkage-induced tyrosine phosphorylation of cortactin. Looking for an alternative signaling mechanism, we found that hypertonicity stimulates Rac and Cdc42. This appears to be a key event in the osmotically triggered cytoskeletal reorganization, because 1) constitutively active small GTPases translocate cortactin, 2) Rac and cortactin colocalize at the periphery of hypertonically challenged cells, and3) dominant-negative Rac and Cdc42 inhibit the hypertonicity-provoked cortactin and Arp3 translocation. The Rho family-dependent cytoskeleton remodeling may be an important osmoprotective response that reinforces the cell cortex.

Epithelial-mesenchymal-myofibroblast transition (EMT), a key feature in organ fibrosis, is regulated by the state of intercellular contacts. Our recent studies have shown that an initial injury of cell–cell junctions is a prerequisite for transforming growth factor-β1 (TGF-β1)-induced transdifferentiation of kidney tubular cells into α-smooth muscle actin (SMA)–expressing myofibroblasts. Here we analyzed the underlying contact-dependent mechanisms. Ca²⁺ removal–induced disruption of intercellular junctions provoked Rho/Rho kinase (ROK)-mediated myosin light chain (MLC) phosphorylation and Rho/ROK-dependent SMA promoter activation. Importantly, myosin-based contractility itself played a causal role, because the myosin ATPase inhibitor blebbistatin or a nonphosphorylatable, dominant negative MLC (DN-MLC) abolished the contact disruption-triggered SMA promoter activation, eliminated the synergy between contact injury and TGF-β1, and suppressed SMA expression. To explore the responsible mechanisms, we investigated the localization of the main SMA-inducing transcription factors, serum response factor (SRF), and its coactivator myocardin-related transcription factor (MRTF). Contact injury enhanced nuclear accumulation of SRF and MRTF. These processes were inhibited by DN-Rho or DN-MLC. TGF-β1 strongly facilitated nuclear accumulation of MRTF in cells with reduced contacts but not in intact epithelia. DN-myocardin abrogated the Ca²⁺-removal– ± TGF-β1–induced promoter activation. These studies define a new mechanism whereby cell contacts regulate epithelial-myofibroblast transition via Rho-ROK-phospho-MLC–dependent nuclear accumulation of MRTF.

Claudin-2 is expressed in the tight junctions of leaky epithelia, where it forms cation-selective and water permeable paracellular channels. Its abundance is under fine control by a complex signaling network that affects both its synthesis and turnover in response to various environmental inputs. Claudin-2 expression is dysregulated in many pathologies including cancer, inflammation, and fibrosis. Claudin-2 has a key role in energy-efficient ion and water transport in the proximal tubules of the kidneys and in the gut. Importantly, strong evidence now also supports a role for this protein as a modulator of vital cellular events relevant to diseases. Signaling pathways that are overactivated in diseases can alter claudin-2 expression, and a good correlation exists between disease stage and claudin-2 abundance. Further, loss- and gain-of-function studies showed that primary changes in claudin-2 expression impact vital cellular processes such as proliferation, migration, and cell fate determination. These effects appear to be mediated by alterations in key signaling pathways. The specific mechanisms linking claudin-2 to these changes remain poorly understood, but adapters binding to the intracellular portion of claudin-2 may play a key role. Thus, dysregulation of claudin-2 may contribute to the generation, maintenance, and/or progression of diseases through both permeability-dependent and -independent mechanisms. The aim of this review is to provide an overview of the properties, regulation, and functions of claudin-2, with a special emphasis on its signal-modulating effects and possible role in diseases.

Objectives: Recently, recombinant angiotensin-converting enzyme 2 was shown to protect mice from acute lung injury, an effect attributed to reduced bioavailability of angiotensin II. Since angiotensin-converting enzyme 2 metabolizes angiotensin II to angiotensin-(1–7), we hypothesized that this effect is alternatively mediated by angiotensin-(1–7) and activation of its receptor(s). Design: To test this hypothesis, we investigated the effects of intravenously infused angiotensin-(1–7) in three experimental models of acute lung injury. Setting: Animal research laboratory. Subjects: Male Sprague-Dawley rats, Balb/c mice, and C57Bl6/J mice. Interventions: Angiotensin-(1–7) was administered with ventilator- or acid aspiration–induced lung injury in mice or 30 minutes after oleic acid infusion in rats. In vitro, the effect of angiotensin-(1–7) on transendothelial electrical resistance of human pulmonary microvascular endothelial cells was analyzed. Measurements and Main Results: Infusion of angiotensin-(1–7) starting 30 minutes after oleic acid administration protected rats from acute lung injury as evident by reduced lung edema, myeloperoxidase activity, histological lung injury score, and pulmonary vascular resistance while systemic arterial pressure was stabilized. Such effects were largely reproduced by the nonpeptidic angiotensin-(1–7) analog AVE0991. Infusion of angiotensin-(1–7) was equally protective in murine models of ventilator- or acid aspiration–induced lung injury. In the oleic acid model, the two distinct angiotensin-(1–7) receptor blockers A779 and D-Pro⁷-angiotensin-(1–7) reversed the normalizing effects of angiotensin-(1–7) on systemic and pulmonary hemodynamics, but only D-Pro⁷-angiotensin-(1–7) blocked the protection from lung edema and protein leak, whereas A779 restored the infiltration of neutrophils. Rats were also protected from acute lung injury by the AT₁ antagonist irbesartan; however, this effect was again blocked by A779 and D-Pro⁷-angiotensin-(1–7). In vitro, angiotensin-(1–7) protected pulmonary microvascular endothelial cells from thrombin-induced barrier failure, yet D-Pro⁷-angiotensin-(1–7) or NO synthase inhibition blocked this effect. Conclusions: Angiotensin-(1–7) or its analogs attenuate the key features of acute lung injury and may present a promising therapeutic strategy for the treatment of this disease.

The epithelial isoform of the Na⁺/H⁺ exchanger, NHE3, associates with at least two related regulatory factors called NHERF1/EBP50 and NHERF2/TKA-1/E3KARP. These factors in addition interact with the cytoskeletal protein ezrin, which in turn binds to actin. The possible linkage of NHE3 with the cytoskeleton prompted us to test the effect of actin-modifying agents on NHE3 activity. Cytochalasins B and D and latrunculin B, which interfere with actin polymerization, induced a profound inhibition of NHE3 activity. The effect was isoform-specific inasmuch as the “housekeeping” exchanger NHE1 was virtually unaffected. Cytoskeletal disorganization was associated with a subcellular redistribution of NHE3, which accumulated at sites where actin aggregated, suggesting a physical interaction of exchangers with the cytoskeleton. An interaction was further suggested by the co-sedimentation of a detergent-insoluble fraction of NHE3 with the actin cytoskeleton. Inhibition of transport was not due to diminution in the number of transporters at the plasmalemma. Functional analyses of NHE1/NHE3 chimeras revealed that the cytoplasmic domain of NHE3 conferred sensitivity to cytochalasin B. Progressive carboxyl-terminal and internal deletions of the cytoplasmic region of NHE3 indicated that the region between residues 650 and 684 is critical for this response. This region overlaps with the domain reported to interact with NHERF and also contains a putative ezrin-binding site; hence, it likely plays a role in interactions with the cytoskeleton.