EphB2 Receptor Promotes Dermal Fibrosis in Systemic Sclerosis.

OBJECTIVE: Erythropoietin-producing hepatocellular (Eph)/Ephrin cell-cell signaling is emerging as a key player in tissue fibrogenesis. The aim of this study was to test the hypothesis that the receptor tyrosine kinase EphB2 mediates dermal fibrosis in systemic sclerosis (SSc). METHODS: We assessed normal and SSc human skin biopsies for EphB2 expression. The in vivo role of EphB2 in skin fibrosis was investigated by subjecting EphB2-knockout mice to both bleomycin-induced and tight skin (Tsk1/+) genetic mouse models of skin fibrosis. EphB2 kinase-dead and overactive point mutant mice were used to evaluate the role of EphB2 forward signaling in bleomycin-induced dermal fibrosis. In vitro studies were performed on dermal fibroblasts from patients with SSc and healthy controls, which was followed by in vivo analysis of fibroblast-specific Ephb2-deficient mice. RESULTS: Expression of EphB2 is up-regulated in SSc skin tissue and explanted SSc dermal fibroblasts compared with healthy controls. EphB2 expression is elevated in two animal models of dermal fibrosis. In mice, EphB2 drives dermal fibrosis in both the bleomycin and the Tsk1/+ models of skin fibrosis. EphB2 forward signaling is a critical mediator of dermal fibrosis. Transforming growth factor-ß (TGF-ß) cytokines up-regulate EphB2 in dermal fibroblasts via noncanonical TGF-ß/mother against decapentaplegic signaling, and silencing EPHB2 in human dermal fibroblasts is sufficient to dampen TGF-ß-induced fibroblast-to-myofibroblast differentiation. Moreover, mice with fibroblast-specific deletion of EphB2 showed impaired fibroblast-to-myofibroblast differentiation and reduced skin fibrosis upon bleomycin challenge. CONCLUSION: Our data implicate TGF-ß regulation of EphB2 overexpression and kinase-mediated forward signaling in the development of dermal fibrosis in SSc. EphB2 thus represents a potential new therapeutic target for SSc.

PIP2 Interacts Electrostatically with MARCKS-like Protein-1 and ENaC in Renal Epithelial Cells.

We examined the interaction of a membrane-associated protein, MARCKS-like Protein-1 (MLP-1), and an ion channel, Epithelial Sodium Channel (ENaC), with the anionic lipid, phosphatidylinositol 4, 5-bisphosphate (PIP2). We found that PIP2 strongly activates ENaC in excised, inside-out patches with a half-activating concentration of 21 ± 1.17 µM. We have identified 2 PIP2 binding sites in the N-terminus of ENaC ß and γ with a high concentration of basic residues. Normal channel activity requires MLP-1's strongly positively charged effector domain to electrostatically sequester most of the membrane PIP2 and increase the local concentration of PIP2. Our previous data showed that ENaC covalently binds MLP-1 so PIP2 bound to MLP-1 would be near PIP2 binding sites on the cytosolic N terminal regions of ENaC. We have modified the charge structure of the PIP2 -binding domains of MLP-1 and ENaC and showed that the changes affect membrane localization and ENaC activity in a way consistent with electrostatic theory.

Genetic analyses in mouse fibroblast and melanoma cells demonstrate novel roles for PDGF-AB ligand and PDGF receptor alpha.

Platelet Derived Growth Factor Receptor (PDGFR) signaling is a central mitogenic pathway in development, as well as tissue repair and homeostasis. The rules governing the binding of PDGF ligand to the receptor to produce activation and downstream signaling have been well defined over the last several decades. In cultured cells after a period of serum deprivation, treatment with PDGF leads to the rapid formation of dramatic, actin-rich Circular Dorsal Ruffles (CDRs). Using CDRs as a robust visual readout of early PDGFR signaling, we have identified several contradictory elements in the widely accepted model of PDGF activity. Employing CRISPR/Cas9 gene editing to disrupt the Pdgfra gene in two different murine cell lines, we show that in addition to the widely accepted function for PDGFR-beta in CDR formation, PDGFR-alpha is also clearly capable of eliciting CDRs. Moreover, we demonstrate activity for heterodimeric PDGF-AB ligand in the vigorous activation of PDGFR-beta homodimers to produce CDRs. These findings are key to a more complete understanding of PDGF ligand-receptor interactions and their downstream signaling consequences. This knowledge will allow for more rigorous experimental design in future studies of PDGFR signaling and its contributions to development and disease.

Mechanical stress triggers nuclear remodeling and the formation of transmembrane actin nuclear lines with associated nuclear pore complexes.

Mechanical stimulation of fibroblasts induces changes in the actin cytoskeleton including stress fiber (SF) reinforcement and realignment. Here we characterize the nuclear response to mechanical stimulation (uniaxial cyclic stretch). Using fluorescence microscopy and quantitative image analysis we find that stretch-induced nuclear elongation and alignment perpendicular to the stretch vector are dependent on formin-regulated actin polymerization. The mechanosensitive transcription factors Yes-associated protein/Transcriptional coactivator with PDZ domain (YAP/TAZ) and myocardin-related transcription factor (MRTF-A, also known as MKL1 and MAL1) accumulate in the nucleus and activate their target genes in response to uniaxial cyclic stretch. We show that transmembrane actin nuclear (TAN) lines are induced by stretch stimulation and nuclear envelope (NE) proteins including nesprins, SUN2, and lamins form Linkers of the Nucleoskeleton and Cytoskeleton (LINC) complexes aligned with actin SFs. These NE structures are altered by pharmacological treatments (Cytochalasin D and Jasplakinolide) or genetic disruption (zyxin gene deletion) that alter actin, and their persistence requires maintenance of stretch stimulation. Nuclear pore complexes (NPCs) accumulate at TAN lines providing a potential mechanism for linking mechanical cues to NPC function.

Noncanonical translation via deadenylated 3' UTRs maintains primordial germ cells.

Primordial germ cells (PGCs) form during early embryogenesis with a supply of maternal mRNAs that contain shorter poly(A) tails. How translation of maternal mRNAs is regulated during PGC development remains elusive. Here we describe a small-molecule screen with zebrafish embryos that identified primordazine, a compound that selectively ablates PGCs. Primordazine's effect on PGCs arises from translation repression through primordazine-response elements in the 3' UTRs. Systematic dissection of primordazine's mechanism of action revealed that translation of mRNAs during early embryogenesis occurs by two distinct pathways, depending on the length of their poly(A) tails. In addition to poly(A)-tail-dependent translation (PAT), early embryos perform poly(A)-tail-independent noncanonical translation (PAINT) via deadenylated 3' UTRs. Primordazine inhibits PAINT without inhibiting PAT, an effect that was also observed in quiescent, but not proliferating, mammalian cells. These studies reveal that PAINT is an alternative form of translation in the early embryo and is indispensable for PGC maintenance.

Mechanical signals activate p38 MAPK pathway-dependent reinforcement of actin via mechanosensitive HspB1.

Despite the importance of a cell's ability to sense and respond to mechanical force, the molecular mechanisms by which physical cues are converted to cell-instructive chemical information to influence cell behaviors remain to be elucidated. Exposure of cultured fibroblasts to uniaxial cyclic stretch results in an actin stress fiber reinforcement response that stabilizes the actin cytoskeleton. p38 MAPK signaling is activated in response to stretch, and inhibition of p38 MAPK abrogates stretch-induced cytoskeletal reorganization. Here we show that the small heat shock protein HspB1 (hsp25/27) is phosphorylated in stretch-stimulated mouse fibroblasts via a p38 MAPK-dependent mechanism. Phosphorylated HspB1 is recruited to the actin cytoskeleton, displaying prominent accumulation on actin "comet tails" that emanate from focal adhesions in stretch-stimulated cells. Site-directed mutagenesis to block HspB1 phosphorylation inhibits the protein's cytoskeletal recruitment in response to mechanical stimulation. HspB1-null cells, generated by CRISPR/Cas9 nuclease genome editing, display an abrogated stretch-stimulated actin reinforcement response and increased cell migration. HspB1 is recruited to sites of increased traction force in cells geometrically constrained on micropatterned substrates. Our findings elucidate a molecular pathway by which a mechanical signal is transduced via activation of p38 MAPK to influence actin remodeling and cell migration via a zyxin-independent process.

Interactions by 2D Gel Electrophoresis Overlap (iGEO): a novel high fidelity approach to identify constituents of protein complexes.

BACKGROUND: Here we describe a novel approach used to identify the constituents of protein complexes with high fidelity, using the integrin-associated scaffolding protein PINCH as a test case. PINCH is comprised of five LIM domains, zinc-finger protein interaction modules. In Drosophila melanogaster, PINCH has two known high-affinity binding partners-Integrin-linked kinase (ILK) that binds to LIM1 and Ras Suppressor 1 (RSU1) that binds to LIM5-but has been postulated to bind additional proteins as well. RESULTS: To purify PINCH complexes, in parallel we fused different affinity tags (Protein A and Flag) to different locations within the PINCH sequence (N- and C-terminus). We expressed these tagged versions of PINCH both in cell culture (overexpressed in Drosophila S2 cell culture in the presence of endogenous PINCH) and in vivo (at native levels in Drosophila lacking endogenous PINCH). After affinity purification, we analyzed PINCH complexes by a novel 2D-gel electrophoresis analysis, iGEO (interactions by 2D Gel Electrophoresis Overlap), with mass spectrometric identification of individual spots of interest. iGEO allowed the identification of protein partners that associate with PINCH under two independent purification strategies, providing confidence in the significance of the interaction. Proteins identified by iGEO were validated against a highly inclusive list of candidate PINCH interacting proteins identified in previous analyses by MuDPIT mass spectrometry. CONCLUSIONS: The iGEO strategy confirmed a core complex comprised of PINCH, RSU1, ILK, and ILK binding partner Parvin. Our iGEO method also identified five novel protein partners that specifically interacted with PINCH in Drosophila S2 cell culture. Because of the improved reproducibility of 2D-GE methodology and the increasing affordability of the required labeling reagents, iGEO is a method that is accessible to most moderately well-equipped biological laboratories. The biochemical co-purifications inherent in iGEO allow for rapid and unambiguous identification of the constituents of protein complexes, without the need for extensive follow-up experiments.

Crowding induces live cell extrusion to maintain homeostatic cell numbers in epithelia.

For an epithelium to provide a protective barrier, it must maintain homeostatic cell numbers by matching the number of dividing cells with the number of dying cells. Although compensatory cell division can be triggered by dying cells, it is unknown how cell death might relieve overcrowding due to proliferation. When we trigger apoptosis in epithelia, dying cells are extruded to preserve a functional barrier. Extrusion occurs by cells destined to die signalling to surrounding epithelial cells to contract an actomyosin ring that squeezes the dying cell out. However, it is not clear what drives cell death during normal homeostasis. Here we show in human, canine and zebrafish cells that overcrowding due to proliferation and migration induces extrusion of live cells to control epithelial cell numbers. Extrusion of live cells occurs at sites where the highest crowding occurs in vivo and can be induced by experimentally overcrowding monolayers in vitro. Like apoptotic cell extrusion, live cell extrusion resulting from overcrowding also requires sphingosine 1-phosphate signalling and Rho-kinase-dependent myosin contraction, but is distinguished by signalling through stretch-activated channels. Moreover, disruption of a stretch-activated channel, Piezo1, in zebrafish prevents extrusion and leads to the formation of epithelial cell masses. Our findings reveal that during homeostatic turnover, growth and division of epithelial cells on a confined substratum cause overcrowding that leads to their extrusion and consequent death owing to the loss of survival factors. These results suggest that live cell extrusion could be a tumour-suppressive mechanism that prevents the accumulation of excess epithelial cells.

Stretch-induced actin remodeling requires targeting of zyxin to stress fibers and recruitment of actin regulators.

Reinforcement of actin stress fibers in response to mechanical stimulation depends on a posttranslational mechanism that requires the LIM protein zyxin. The C-terminal LIM region of zyxin directs the force-sensitive accumulation of zyxin on actin stress fibers. The N-terminal region of zyxin promotes actin reinforcement even when Rho kinase is inhibited. The mechanosensitive integrin effector p130Cas binds zyxin but is not required for mitogen-activated protein kinase-dependent zyxin phosphorylation or stress fiber remodeling in cells exposed to uniaxial cyclic stretch. α-Actinin and Ena/VASP proteins bind to the stress fiber reinforcement domain of zyxin. Mutation of their docking sites reveals that zyxin is required for recruitment of both groups of proteins to regions of stress fiber remodeling. Zyxin-null cells reconstituted with zyxin variants that lack either α-actinin or Ena/VASP-binding capacity display compromised response to mechanical stimulation. Our findings define a bipartite mechanism for stretch-induced actin remodeling that involves mechanosensitive targeting of zyxin to actin stress fibers and localized recruitment of actin regulatory machinery.

ß3 integrin-EGF receptor cross-talk activates p190RhoGAP in mouse mammary gland epithelial cells.

Active RhoA localizes to plasma membrane, where it stimulates formation of focal adhesions and stress fibers. RhoA activity is inhibited by p190RhoGAP following integrin-mediated cell attachment to allow sampling of new adhesive environments. p190RhoGAP is itself activated by Src-dependent tyrosine phosphorylation, which facilitates complex formation with p120RasGAP. This complex then translocates to the cell surface, where p190RhoGAP down-regulates RhoA. Here we demonstrate that the epidermal growth factor receptor (EGFR) cooperates with ß3 integrin to regulate p190RhoGAP activity in mouse mammary gland epithelial cells. Adhesion to fibronectin stimulates tyrosine phosphorylation of the EGFR in the absence of receptor ligands. Use of a dominant inhibitory EGFR mutant demonstrates that fibronectin-activated EGFR recruits p120RasGAP to the cell periphery. Expression of an inactive ß3 integrin subunit abolishes p190RhoGAP tyrosine phosphorylation, demonstrating a mechanistic link between ß3 integrin-activated Src and EGFR regulation of the RhoA inhibitor. The ß3 integrin/EGFR pathway also has a positive role in formation of filopodia. Together our data suggest that EGFR constitutes an important intrinsic migratory cue since fibronectin is a key component of the microenvironment in normal mammary gland development and breast cancer. Our data also suggest that EGFR expressed at high levels has a role in eliciting cell shape changes associated with epithelial-to-mesenchymal transition.

Loss of the serum response factor cofactor, cysteine-rich protein 1, attenuates neointima formation in the mouse.

OBJECTIVE: Cysteine-rich protein (CRP) 1 and 2 are cytoskeletal lin-11 isl-1 mec-3 (LIM)-domain proteins thought to be critical for smooth muscle differentiation. Loss of murine CRP2 does not overtly affect smooth muscle differentiation or vascular function but does exacerbate neointima formation in response to vascular injury. Because CRPs 1 and 2 are coexpressed in the vasculature, we hypothesize that CRPs 1 and 2 act redundantly in smooth muscle differentiation. METHODS AND RESULTS: We generated Csrp1 (gene name for CRP1) null mice by genetic ablation of the Csrp1 gene and found that mice lacking CRP1 are viable and fertile. Smooth muscle-containing tissues from Csrp1-null mice are morphologically indistinguishable from wild-type mice and have normal contractile properties. Mice lacking CRPs 1 and 2 are viable and fertile, ruling out functional redundancy between these 2 highly related proteins as a cause for the lack of an overt phenotype in the Csrp1-null mice. Csrp1-null mice challenged by wire-induced arterial injury display reduced neointima formation, opposite to that seen in Csrp2-null mice, whereas Csrp1/Csrp2 double-null mice produce a wild-type response. CONCLUSIONS: Smooth muscle CRPs are not essential for normal smooth muscle differentiation during development, but may act antagonistically to modulate the smooth muscle response to pathophysiological stress.

Genetic ablation of zyxin causes Mena/VASP mislocalization, increased motility, and deficits in actin remodeling.

Focal adhesions are specialized regions of the cell surface where integrin receptors and associated proteins link the extracellular matrix to the actin cytoskeleton. To define the cellular role of the focal adhesion protein zyxin, we characterized the phenotype of fibroblasts in which the zyxin gene was deleted by homologous recombination. Zyxin-null fibroblasts display enhanced integrin-dependent adhesion and are more migratory than wild-type fibroblasts, displaying reduced dependence on extracellular matrix cues. We identified differences in the profiles of 75- and 80-kD tyrosine-phosphorylated proteins in the zyxin-null cells. Tandem array mass spectrometry identified both modified proteins as isoforms of the actomyosin regulator caldesmon, a protein known to influence contractility, stress fiber formation, and motility. Zyxin-null fibroblasts also show deficits in actin stress fiber remodeling and exhibit changes in the molecular composition of focal adhesions, most notably by severely reduced accumulation of Ena/VASP proteins. We postulate that zyxin cooperates with Ena/VASP proteins and caldesmon to influence integrin-dependent cell motility and actin stress fiber remodeling.

Mechanical force mobilizes zyxin from focal adhesions to actin filaments and regulates cytoskeletal reinforcement.

Organs and tissues adapt to acute or chronic mechanical stress by remodeling their actin cytoskeletons. Cells that are stimulated by cyclic stretch or shear stress in vitro undergo bimodal cytoskeletal responses that include rapid reinforcement and gradual reorientation of actin stress fibers; however, the mechanism by which cells respond to mechanical cues has been obscure. We report that the application of either unidirectional cyclic stretch or shear stress to cells results in robust mobilization of zyxin from focal adhesions to actin filaments, whereas many other focal adhesion proteins and zyxin family members remain at focal adhesions. Mechanical stress also induces the rapid zyxin-dependent mobilization of vasodilator-stimulated phosphoprotein from focal adhesions to actin filaments. Thickening of actin stress fibers reflects a cellular adaptation to mechanical stress; this cytoskeletal reinforcement coincides with zyxin mobilization and is abrogated in zyxin-null cells. Our findings identify zyxin as a mechanosensitive protein and provide mechanistic insight into how cells respond to mechanical cues.

Quantification of stretch-induced cytoskeletal remodeling in vascular endothelial cells by image processing.

BACKGROUND: Reorientation of the cell axis induced by cyclic stretching is an early response to mechanical forces in vitro. However, quantitative assay for this phenomenon has been difficult due to lack of robust methods. We hypothesized that cell orientation may be redefined by the orientation of actin fibers. We developed image processing methods to quantitate the orientation and density of actin fibers. METHODS: A convolution filter using Sobel kernels was adapted to determine the orientation and density of actin fibers in human endothelial cells. Unidirectional stretching (10%, 0.5 Hz) was applied to induce cytoskeletal remodeling by varying the duration of stimulation (control, 0.5, 1, 2, 5, 10, and 20 h). Actin fibers were visualized by fluorescent phalloidin. The image processing method was compared with the manual method for reproducibility. Both confluent and subconfluent cells were tested to assess the efficacy of the methods. RESULTS: Cyclic stretch-induced dense and uninterrupted actin cabling formed across the cell body and, later, the actin fibers became aligned perpendicular to the stretch direction. The variance of actin fiber orientation became smaller after 2 h of stretch (F < 0.01). The actin fiber density index, a derived parameter related to the density of actin fibers, increased as early as 30 min of stretching (P < 0.05) and decreased after 10 h of stretching. Reproducibility of our method was extremely good. Applicability of the method was not compromised by cell density. CONCLUSIONS: Our method is reliable for quantifying cytoskeletal remodeling induced by mechanical force.