MRCK: a master regulator of tissue remodeling or another 'ROCK' in the epithelial block?

The epithelium forms a smart barrier to the external environment that can remodel whilst maintaining tissue integrity, a feature important for development, homeostasis, and function. Its dysregulation can lead to diseases ranging from cancer to vision loss. Epithelial remodeling requires reorganization of a thin sheet of actomyosin cortex under the plasma membrane of polarized cells that form basolateral contacts with neighboring cells and the extracellular matrix (ECM). Rho GTPases act as spatiotemporal molecular switches in this process, controlling localized actomyosin dynamics. However, the molecular mechanisms that control actomyosin dynamics at the apical cortex are poorly understood. This review focusses on a growing body of evidence that suggest myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) plays a conserved role in morphogenetic signaling at the apical cortex in diverse cell and tissue remodeling processes. The possible molecular and mechanistic basis for the diverse functions of MRCK at the apical pole will also be discussed

Dbl3 drives Cdc42 signaling at the apical margin to regulate junction position and apical differentiation

Epithelial cells develop morphologically characteristic apical domains that are bordered by tight junctions, the apical–lateral border. Cdc42 and its effector complex Par6–atypical protein kinase c (aPKC) regulate multiple steps during epithelial differentiation, but the mechanisms that mediate process-specific activation of Cdc42 to drive apical morphogenesis and activate the transition from junction formation to apical differentiation are poorly understood. Using a small interfering RNA screen, we identify Dbl3 as a guanine nucleotide exchange factor that is recruited by ezrin to the apical membrane, that is enriched at a marginal zone apical to tight junctions, and that drives spatially restricted Cdc42 activation, promoting apical differentiation. Dbl3 depletion did not affect junction formation but did affect epithelial morphogenesis and brush border formation. Conversely, expression of active Dbl3 drove process-specific activation of the Par6–aPKC pathway, stimulating the transition from junction formation to apical differentiation and domain expansion, as well as the positioning of tight junctions. Thus, Dbl3 drives Cdc42 signaling at the apical margin to regulate morphogenesis, apical–lateral border positioning, and apical differentiation

Stimulation of cortical myosin phosphorylation by p114RhoGEF drives cell migration and tumor cell invasion.

Actinomyosin activity is an important driver of cell locomotion and has been shown to promote collective cell migration of epithelial sheets as well as single cell migration and tumor cell invasion. However, the molecular mechanisms underlying activation of cortical myosin to stimulate single cell movement, and the relationship between the mechanisms that drive single cell locomotion and those that mediate collective cell migration of epithelial sheets are incompletely understood. Here, we demonstrate that p114RhoGEF, an activator of RhoA that associates with non-muscle myosin IIA, regulates collective cell migration of epithelial sheets and tumor cell invasion. Depletion of p114RhoGEF resulted in specific spatial inhibition of myosin activation at cell-cell contacts in migrating epithelial sheets and the cortex of migrating single cells, but only affected double and not single phosphorylation of myosin light chain. In agreement, overall elasticity and contractility of the cells, processes that rely on persistent and more constant forces, were not affected, suggesting that p114RhoGEF mediates process-specific myosin activation. Locomotion was p114RhoGEF-dependent on Matrigel, which favors more roundish cells and amoeboid-like actinomyosin-driven movement, but not on fibronectin, which stimulates flatter cells and lamellipodia-driven, mesenchymal-like migration. Accordingly, depletion of p114RhoGEF led to reduced RhoA, but increased Rac activity. Invasion of 3D matrices was p114RhoGEF-dependent under conditions that do not require metalloproteinase activity, supporting a role of p114RhoGEF in myosin-dependent, amoeboid-like locomotion. Our data demonstrate that p114RhoGEF drives cortical myosin activation by stimulating myosin light chain double phosphorylation and, thereby, collective cell migration of epithelial sheets and amoeboid-like motility of tumor cells

Interstitial cystitis: a rare manifestation of primary Sjögren’s syndrome, successfully treated with low dose cyclosporine

Chronic interstitial cystitis (IC), mostly affecting middle-aged women, is a very rare manifestation of primary Sjögren’s syndrome (pSS). Hereby, we report a 42-year-old woman with pSS, presenting with dysuria, urinary frequency, and suprapubic pain. She was diagnosed to have chronic IC, based upon the cystoscopic biopsy finding of chronic inflammation in the bladder wall. Systemic corticosteroid and azathioprine treatments together with local intravesical therapies were not effective. Therefore, cyclosporine (CSA) therapy was initiated. Initial low dose of CSA (1.5 mg/kg/d) improved the symptoms of the patient, with no requirement for dose increment. After 4 months of therapy, control cystoscopic biopsy showed that bladder inflammation regressed and IC improved. This case suggests that even low doses of CSA may be beneficial for treating chronic IC associated with pSS syndrome

The Role of Purported Mucoprotectants in Dealing with Irritable Bowel Syndrome, Functional Diarrhea, and Other Chronic Diarrheal Disorders in Adults

Chronic diarrhea is a frequent presenting symptom, both in primary care medicine and in specialized gastroenterology units. It is estimated that more than 5% of the global population suffers from chronic diarrhea. and that about 40% of these subjects are older than 60 years. The clinician is frequently faced with the need to decide which is the best therapeutic approach for these patients. While the origin of chronic diarrhea is diverse, impairment of intestinal barrier function, dysbiosis. and mucosal micro-inflammation are being increasingly recognized as underlying phenomena characterizing a variety of chronic diarrheal diseases. In addition to current pharmacological therapies, there is growing interest in alternative products such as mucoprotectants, which form a mucoadhesive film over the epithelium to reduce and protect against the development of altered intestinal permeability, dysbiosis, and mucosal micro-inflammation. This manuscript focuses on chronic diarrhea in adults, and we will review recent evidence on the ability of these natural compounds to improve symptoms associated with chronic diarrhea and to exert protective effects for the intestinal barrier

A Role for Thrombospondin-1 Deficits in Astrocyte-Mediated Spine and Synaptic Pathology in Down's Syndrome

Down's syndrome (DS) is the most common genetic cause of mental retardation. Reduced number and aberrant architecture of dendritic spines are common features of DS neuropathology. However, the mechanisms involved in DS spine alterations are not known. In addition to a relevant role in synapse formation and maintenance, astrocytes can regulate spine dynamics by releasing soluble factors or by physical contact with neurons. We have previously shown impaired mitochondrial function in DS astrocytes leading to metabolic alterations in protein processing and secretion. In this study, we investigated whether deficits in astrocyte function contribute to DS spine pathology.Using a human astrocyte/rat hippocampal neuron coculture, we found that DS astrocytes are directly involved in the development of spine malformations and reduced synaptic density. We also show that thrombospondin 1 (TSP-1), an astrocyte-secreted protein, possesses a potent modulatory effect on spine number and morphology, and that both DS brains and DS astrocytes exhibit marked deficits in TSP-1 protein expression. Depletion of TSP-1 from normal astrocytes resulted in dramatic changes in spine morphology, while restoration of TSP-1 levels prevented DS astrocyte-mediated spine and synaptic alterations. Astrocyte cultures derived from TSP-1 KO mice exhibited similar deficits to support spine formation and structure than DS astrocytes.These results indicate that human astrocytes promote spine and synapse formation, identify astrocyte dysfunction as a significant factor of spine and synaptic pathology in the DS brain, and provide a mechanistic rationale for the exploration of TSP-1-based therapies to treat spine and synaptic pathology in DS and other neurological conditions

MarvelD3 couples tight junctions to the MEKK1-JNK pathway to regulate cell behavior and survival

MarvelD3 is a transmembrane component of tight junctions, but there is little evidence for a direct involvement in the junctional permeability barrier. Tight junctions also regulate signaling mechanisms that guide cell proliferation; however, the transmembrane components that link the junction to such signaling pathways are not well understood. In this paper, we show that MarvelD3 is a dynamic junctional regulator of the MEKK1-c-Jun NH2-terminal kinase (JNK) pathway. Loss of MarvelD3 expression in differentiating Caco-2 cells resulted in increased cell migration and proliferation, whereas reexpression in a metastatic tumor cell line inhibited migration, proliferation, and in vivo tumor formation. Expression levels of MarvelD3 inversely correlated with JNK activity, as MarvelD3 recruited MEKK1 to junctions, leading to down-regulation of JNK phosphorylation and inhibition of JNK-regulated transcriptional mechanisms. Interplay between MarvelD3 internalization and JNK activation tuned activation of MEKK1 during osmotic stress, leading to junction dissociation and cell death in MarvelD3-depleted cells. MarvelD3 thus couples tight junctions to the MEKK1-JNK pathway to regulate cell behavior and survival

Low-dose acetaminophen induces early disruption of cell-cell tight junctions in human hepatic cells and mouse liver

Dysfunction of cell-cell tight junction (TJ) adhesions is a major feature in the pathogenesis of various diseases. Liver TJs preserve cellular polarity by delimiting functional bile-canalicular structures, forming the blood-biliary barrier. In acetaminophen-hepatotoxicity, the mechanism by which tissue cohesion and polarity are affected remains unclear. Here, we demonstrate that acetaminophen, even at low-dose, disrupts the integrity of TJ and cell-matrix adhesions, with indicators of cellular stress with liver injury in the human hepatic HepaRG cell line, and primary hepatocytes. In mouse liver, at human-equivalence (therapeutic) doses, dose-dependent loss of intercellular hepatic TJ-associated ZO-1 protein expression was evident with progressive clinical signs of liver injury. Temporal, dose-dependent and specific disruption of the TJ-associated ZO-1 and cytoskeletal-F-actin proteins, correlated with modulation of hepatic ultrastructure. Real-time impedance biosensing verified in vitro early, dose-dependent quantitative decreases in TJ and cell-substrate adhesions. Whereas treatment with NAPQI, the reactive metabolite of acetaminophen, or the PKCα-activator and TJ-disruptor phorbol-12-myristate-13-acetate, similarly reduced TJ integrity, which may implicate oxidative stress and the PKC pathway in TJ destabilization. These findings are relevant to the clinical presentation of acetaminophen-hepatotoxicity and may inform future mechanistic studies to identify specific molecular targets and pathways that may be altered in acetaminophen-induced hepatic depolarization

Tight junctions: from simple barriers to multifunctional molecular gates

Epithelia and endothelia separate different tissue compartments and protect multicellular organisms from the outside world. This requires the formation of tight junctions, selective gates that control paracellular diffusion of ions and solutes. Tight junctions also form the border between the apical and basolateral plasma-membrane domains and are linked to the machinery that controls apicobasal polarization. Additionally, signalling networks that guide diverse cell behaviours and functions are connected to tight junctions, transmitting information to and from the cytoskeleton, nucleus and different cell adhesion complexes. Recent advances have broadened our understanding of the molecular architecture and cellular functions of tight junctions

Biallelic Mutation of ARHGEF18, Involved in the Determination of Epithelial Apicobasal Polarity, Causes Adult-Onset Retinal Degeneration

Mutations in more than 250 genes are implicated in inherited retinal dystrophy; the encoded proteins are involved in a broad spectrum of pathways. The presence of unsolved families after highly parallel sequencing strategies suggests that further genes remain to be identified. Whole-exome and -genome sequencing studies employed here in large cohorts of affected individuals revealed biallelic mutations in ARHGEF18 in three such individuals. ARHGEF18 encodes ARHGEF18, a guanine nucleotide exchange factor that activates RHOA, a small GTPase protein that is a key component of tight junctions and adherens junctions. This biological pathway is known to be important for retinal development and function, as mutation of CRB1, encoding another component, causes retinal dystrophy. The retinal structure in individuals with ARHGEF18 mutations resembled that seen in subjects with CRB1 mutations. Five mutations were found on six alleles in the three individuals: c.808A>G (p.Thr270Ala), c.1617+5G>A (p.Asp540Glyfs∗63), c.1996C>T (p.Arg666∗), c.2632G>T (p.Glu878∗), and c.2738_2761del (p.Arg913_Glu920del). Functional tests suggest that each disease genotype might retain some ARHGEF18 activity, such that the phenotype described here is not the consequence of nullizygosity. In particular, the p.Thr270Ala missense variant affects a highly conserved residue in the DBL homology domain, which is required for the interaction and activation of RHOA. Previously, knock-out of Arhgef18 in the medaka fish has been shown to cause larval lethality which is preceded by retinal defects that resemble those seen in zebrafish Crumbs complex knock-outs. The findings described here emphasize the peculiar sensitivity of the retina to perturbations of this pathway, which is highlighted as a target for potential therapeutic strategies