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The mechanisms underlying diarrhea-predominant irritable bowel syndrome (IBS-D) are poorly understood, but increased intestinal permeability is thought to contribute to symptoms. A recent clinical trial of gluten-free diet (GFD) demonstrated symptomatic improvement, relative to gluten-containing diet (GCD), which was associated with reduced intestinal permeability in non-celiac disease IBS-D patients. The aim of this study was to characterize intestinal epithelial tight junction composition in IBS-D before and after dietary gluten challenge. Biopsies from 27 IBS-D patients (13 GFD and 14 GCD) were examined by H&E staining and semiquantitative immunohistochemistry for phosphorylated myosin II regulatory light chain (MLC), MLC kinase, claudin-2, claudin-8 and claudin-15. Diet-induced changes were assessed and correlated with urinary mannitol excretion (after oral administration). In the small intestine, epithelial MLC phosphorylation was increased or decreased by GCD or GFD, respectively, and this correlated with increased intestinal permeability (P<0.03). Colonocyte expression of the paracellular Na+ channel claudin-15 was also markedly augmented following GCD challenge (P<0.05). Conversely, colonic claudin-2 expression correlated with reduced intestinal permeability (P<0.03). Claudin-8 expression was not affected by dietary challenge. These data show that alterations in MLC phosphorylation and claudin-15 and claudin-2 expression are associated with gluten-induced symptomatology and intestinal permeability changes in IBS-D. The results provide new insight into IBS-D mechanisms and can explain permeability responses to gluten challenge in these patients.

The intestinal epithelial tight junction (TJ) barrier controls the paracellular permeation of contents from the intestinal lumen into the intestinal tissue and systemic circulation. A defective intestinal TJ barrier has been implicated as an important pathogenic factor in inflammatory diseases of the gut including Crohn’s disease, ulcerative colitis, necrotizing enterocolitis, and celiac disease. Previous studies have shown that pro-inflammatory cytokines, which are produced during intestinal inflammation, including interleukin-1β (IL-1β), tumor necrosis factor-α, and interferon-γ, have important intestinal TJ barrier-modulating actions. Recent studies have shown that the IL-1β-induced increase in intestinal TJ permeability is an important contributing factor of intestinal inflammation. The IL-1β-induced increase in intestinal TJ permeability is mediated by regulatory signaling pathways and activation of nuclear transcription factor nuclear factor-κB, myosin light chain kinase gene activation, and post-transcriptional occludin gene modulation by microRNA and contributes to the intestinal inflammatory process. In this review, the regulatory role of IL-1β on intestinal TJ barrier, the intracellular mechanisms that mediate the IL-1β modulation of intestinal TJ permeability, and the potential therapeutic targeting of the TJ barrier are discussed.

Epithelial barrier loss is a driver of intestinal and systemic diseases. Myosin light chain kinase (MLCK) is a key effector of barrier dysfunction and a potential therapeutic target, but enzymatic inhibition has unacceptable toxicity. Here, we show that a unique domain within the MLCK splice variant MLCK1 directs perijunctional actomyosin ring (PAMR) recruitment. Using the domain structure and multiple screens, we identify a domain-binding small molecule (divertin) that blocks MLCK1 recruitment without inhibiting enzymatic function. Divertin blocks acute, tumor necrosis factor (TNF)-induced MLCK1 recruitment as well as downstream myosin light chain (MLC) phosphorylation, barrier loss, and diarrhea in vitro and in vivo. Divertin corrects barrier dysfunction and prevents disease development and progression in experimental inflammatory bowel disease. Beyond applications of divertin in gastrointestinal disease, this general approach to enzymatic inhibition by preventing access to specific subcellular sites provides a new paradigm for safely and precisely targeting individual properties of enzymes with multiple functions. A small molecule that restores the integrity of the intestinal barrier provides a novel therapeutic strategy for inflammatory bowel diseases.

As a physiological small molecular product from the microbial fermentation of dietary fibers, butyrate plays an important role in maintaining intestinal health. Our previous works have proved that the effect of sodium butyrate (NaB) on the intestinal barrier function is mediated by activation of AMP-activated protein kinase (AMPK). However, the detailed pathway involved remains unknown. Using the calcium switch assay in the Caco-2 cell monolayer model, we found here that NaB activated AMPK mainly by increasing the calcium level, but not the ATP concentration, via promoting store-operated calcium entry (SOCE). Upon the activation of AMPK, NaB promoted the reassembly of tight junctions (TJs) based on reducing the phosphorylation of myosin II regulatory light chain (MLC2) at Ser19 and increasing phosphorylation of protein kinase C β2 (PKCβ2) at Ser660. Inhibiting (protein kinase C β) PKCβ blocked the reassembly of TJs induced by NaB in the barrier monolayer model. These results indicated that NaB could activate the calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) pathway to mediate AMPK phosphorylating, which then inhibited the phosphorylation of MLC2 and promoted the phosphorylation of PKCβ2, respectively, so that the downstream molecules of AMPK coordinately contributed to the reassembly of TJs in the Caco-2 barrier model. These results suggested a potential mechanism of butyrate for intestine homeostasis and protection.

SARS-CoV-2 is the causative agent behind the COVID-19 pandemic, responsible for over 170 million infections, and over 3.7 million deaths worldwide. Efforts to test, treat and vaccinate against this pathogen all benefit from an improved understanding of the basic biology of SARS-CoV-2. Both viral and cellular proteases play a crucial role in SARS-CoV-2 replication. Here, we study proteolytic cleavage of viral and cellular proteins in two cell line models of SARS-CoV-2 replication using mass spectrometry to identify protein neo-N-termini generated through protease activity. We identify previously unknown cleavage sites in multiple viral proteins, including major antigens S and N: the main targets for vaccine and antibody testing efforts. We discover significant increases in cellular cleavage events consistent with cleavage by SARS-CoV-2 main protease, and identify 14 potential high-confidence substrates of the main and papain-like proteases. We show that siRNA depletion of these cellular proteins inhibits SARS-CoV-2 replication, and that drugs targeting two of these proteins: the tyrosine kinase SRC and Ser/Thr kinase MYLK, show a dose-dependent reduction in SARS-CoV-2 titres. Overall, our study provides a powerful resource to understand proteolysis in the context of viral infection, and to inform the development of targeted strategies to inhibit SARS-CoV-2 and treat COVID-19. During SARS-CoV-2 replication, viral and cellular proteases play crucial roles and have been shown to be promising anti-viral targets. Here, Meyer et al. apply mass spectrometry to characterize the proteolytic cleavage profile of viral and cellular proteins in vitro.

The intestinal epithelial apical junctional complex, which includes tight and adherens junctions, contributes to the intestinal barrier function via their role in regulating paracellular permeability. Myosin light chain II (MLC-2), has been shown to be a critical regulatory protein in altering paracellular permeability during gastrointestinal disorders. Previous studies have demonstrated that phosphorylation of MLC-2 is a biochemical marker for perijunctional actomyosin ring contraction, which increases paracellular permeability by regulating the apical junctional complex. The phosphorylation of MLC-2 is dominantly regulated by myosin light chain kinase- (MLCK-) and Rho-associated coiled-coil containing protein kinase- (ROCK-) mediated pathways. In this review, we aim to summarize the current state of knowledge regarding the role of MLCK- and ROCK-mediated pathways in the regulation of the intestinal barrier during normal homeostasis and digestive diseases. Additionally, we will also suggest potential therapeutic targeting of MLCK- and ROCK-associated pathways in gastrointestinal disorders that compromise the intestinal barrier.

Vascular smooth muscle tone is controlled by a balance between the cellular signaling pathways that mediate the generation of force (vasoconstriction) and release of force (vasodilation). The initiation of force is associated with increases in intracellular calcium concentrations, activation of myosin light-chain kinase, increases in the phosphorylation of the regulatory myosin light chains, and actin-myosin crossbridge cycling. There are, however, several signaling pathways modulating Ca 2+ mobilization and Ca 2+ sensitivity of the contractile machinery that secondarily regulate the contractile response of vascular smooth muscle to receptor agonists. Among these regulatory mechanisms involved in the physiological regulation of vascular tone are the cyclic nucleotides (cAMP and cGMP), which are considered the main messengers that mediate vasodilation under physiological conditions. At least four distinct mechanisms are currently thought to be involved in the vasodilator effect of cyclic nucleotides and their dependent protein kinases: (1) the decrease in cytosolic calcium concentration ([Ca 2+ ]c), (2) the hyperpolarization of the smooth muscle cell membrane potential, (3) the reduction in the sensitivity of the contractile machinery by decreasing the [Ca 2+ ]c sensitivity of myosin light-chain phosphorylation, and (4) the reduction in the sensitivity of the contractile machinery by uncoupling contraction from myosin light-chain phosphorylation. This review focuses on each of these mechanisms involved in cyclic nucleotide-dependent relaxation of vascular smooth muscle under physiological conditions.

Myosin regulatory light chain (LC20) phosphorylation plays an important role in vascular smooth muscle contraction and cell migration. Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates LC20 (its only known substrate) exclusively at S19. Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in the regulation of LC20 phosphorylation via direct phosphorylation of LC20 at T18 and S19 and indirectly via phosphorylation of MYPT1 (the myosin targeting subunit of myosin light chain phosphatase, MLCP) and Par-4 (prostate-apoptosis response-4). Phosphorylation of MYPT1 at T696 and T853 inhibits MLCP activity whereas phosphorylation of Par-4 at T163 disrupts its interaction with MYPT1, exposing the sites of phosphorylation in MYPT1 and leading to MLCP inhibition. To evaluate the roles of MLCK, ROCK and ZIPK in these phosphorylation events, we investigated the time courses of phosphorylation of LC20, MYPT1 and Par-4 in serum-stimulated human vascular smooth muscle cells (from coronary and umbilical arteries), and examined the effects of siRNA-mediated MLCK, ROCK and ZIPK knockdown and pharmacological inhibition on these phosphorylation events. Serum stimulation induced rapid phosphorylation of LC20 at T18 and S19, MYPT1 at T696 and T853, and Par-4 at T163, peaking within 30-120 s. MLCK knockdown or inhibition, or Ca2+ chelation with EGTA, had no effect on serum-induced LC20 phosphorylation. ROCK knockdown decreased the levels of phosphorylation of LC20 at T18 and S19, of MYPT1 at T696 and T853, and of Par-4 at T163, whereas ZIPK knockdown decreased LC20 diphosphorylation, but increased phosphorylation of MYPT1 at T696 and T853 and of Par-4 at T163. ROCK inhibition with GSK429286A reduced serum-induced phosphorylation of LC20 at T18 and S19, MYPT1 at T853 and Par-4 at T163, while ZIPK inhibition by HS38 reduced only LC20 diphosphorylation. We also demonstrated that serum stimulation induced phosphorylation (activation) of ZIPK, which was inhibited by ROCK and ZIPK down-regulation and inhibition. Finally, basal phosphorylation of LC20 in the absence of serum stimulation was unaffected by MLCK, ROCK or ZIPK knockdown or inhibition. We conclude that: (i) serum stimulation of cultured human arterial smooth muscle cells results in rapid phosphorylation of LC20, MYPT1, Par-4 and ZIPK, in contrast to the slower phosphorylation of kinases and other proteins involved in other signaling pathways (Akt, ERK1/2, p38 MAPK and HSP27), (ii) ROCK and ZIPK, but not MLCK, are involved in serum-induced phosphorylation of LC20, (iii) ROCK, but not ZIPK, directly phosphorylates MYPT1 at T853 and Par-4 at T163 in response to serum stimulation, (iv) ZIPK phosphorylation is enhanced by serum stimulation and involves phosphorylation by ROCK and autophosphorylation, and (v) basal phosphorylation of LC20 under serum-free conditions is not attributable to MLCK, ROCK or ZIPK.

A majority (~95%) of the gas-exchange surface area is generated through septa formation during alveologenesis. Disruption of this process leads to alveolar simplification and bronchopulmonary dysplasia (BPD), a prevalent disorder in premature infants. Although several models have been proposed, the mechanism of septa formation remains under debate. Here we show that inactivation of myosin light chain kinase (MLCK), a key factor required for myofibroblast contraction, disrupted septa formation, supporting the myofibroblast contraction model of alveologenesis. The alveoli simplification phenotype was accompanied by decreased yes-associated protein (YAP), a key effector in the Hippo mechanotransduction pathway. Expression of activated YAP in Mlck-mutant lungs led to partial reversal of alveolar simplification. In the adult, although Mlck inactivation did not lead to simplification, it prevented reseptation during compensatory regrowth in the pneumonectomy model. These findings revealed that myofibroblast reactivation and contraction are requisite steps toward regenerating the gas-exchange surface in diseases such as BPD and chronic obstructive pulmonary disease (COPD).

Phenotypic polarization of macrophages is regulated by a milieu of cues in the local tissue microenvironment. Although much is known about how soluble factors influence macrophage polarization, relatively little is known about how physical cues present in the extracellular environment might modulate proinflammatory (M1) vs. prohealing (M2) activation. Specifically, the role of cell shape has not been explored, even though it has been observed that macrophages adopt different geometries in vivo. We and others observed that macrophages polarized toward different phenotypes in vitro exhibit dramatic changes in cell shape: M2 cells exhibit an elongated shape compared with M1 cells. Using a micropatterning approach to control macrophage cell shape directly, we demonstrate here that elongation itself, without exogenous cytokines, leads to the expression of M2 phenotype markers and reduces the secretion of inflammatory cytokines. Moreover, elongation enhances the effects of M2-inducing cytokines IL-4 and IL-13 and protects cells from M1-inducing stimuli LPS and IFN-γ. In addition shape- but not cytokine-induced polarization is abrogated when actin and actin/myosin contractility are inhibited by pharmacological agents, suggesting a role for the cytoskeleton in the control of macrophage polarization by cell geometry. Our studies demonstrate that alterations in cell shape associated with changes in ECM architecture may provide integral cues to modulate macrophage phenotype polarization.