Audrey M. Bernstein

FAK-dependent regulation of myofibroblast differentiation

Fibroblasts and myofibroblasts both participate in wound healing. Transforming growth factor beta (TGFβ) induces fibroblasts to differentiate into myofibroblasts, whereas fibroblast growth factor and heparin (FGF/h) induce myofibroblasts to “de‐differentiate” into fibroblasts. TGFβ induces expression of smooth muscle alpha actin (SMαA) and incorporation into in stress fibers, a phenotype of differentiated myofibroblasts. Additionally, TGFβ induces the expression of fibronectin and fibronectin integrins. Fibronectin‐generated signals contribute to the TGFβ‐mediated myofibroblast differentiation. Because fibronectin signals are transmitted through focal adhesion kinase (FAK), it was predicted that FAK would be essential to TGFβ‐mediated myofibroblast differentiation. To determine whether the FAK signaling pathway is required for myofibroblast differentiation, we used two approaches to decrease FAK in mouse embryo fibroblasts (MEFs): 1) FAK +/+ MEFs, in which FAK protein expression was greatly decreased by short hairpin RNA (shRNA), and 2) FAK −/− MEFs, which lack FAK. In both cases, the majority of cells were myofibroblasts, expressing SMαA in stress fibers even after treatment with FGF/h. Furthermore, both the surface expression of FGFRs and FGF signaling were greatly reduced in FAK−/− MEFs. We conclude that FAK does not contribute to TGFβ‐dependent myofibroblast differentiation. Instead, FAK was necessary for FGF/h signaling in down‐regulating expression of SMαA, which is synonymous with myofibroblast differentiation. FAK activation could contribute to regulating myofibroblast differentiation, thereby ameliorating fibrosis.—Greenberg, R. S., Bernstein, A. M., Benezra, M., Gelman, I. H., Taliana, L., Masur, S. K. FAK‐dependent regulation of myofibroblast differentiation. FASEB J. 20, E191–E200 (2006)

Mutations in PDGFRB cause autosomal-dominant infantile myofibromatosis.

Infantile myofibromatosis (IM) is a disorder of mesenchymal proliferation characterized by the development of nonmetastasizing tumors in the skin, muscle, bone, and viscera. Occurrence within families across multiple generations is suggestive of an autosomal-dominant (AD) inheritance pattern, but autosomal-recessive (AR) modes of inheritance have also been proposed. We performed whole-exome sequencing (WES) in members of nine unrelated families clinically diagnosed with AD IM to identify the genetic origin of the disorder. In eight of the families, we identified one of two disease-causing mutations, c.1978C>A (p.Pro660Thr) and c.1681C>T (p.Arg561Cys), in PDGFRB. Intriguingly, one family did not have either of these PDGFRB mutations but all affected individuals had a c.4556T>C (p.Leu1519Pro) mutation in NOTCH3. Our studies suggest that mutations in PDGFRB are a cause of IM and highlight NOTCH3 as a candidate gene. Further studies of the crosstalk between PDGFRB and NOTCH pathways may offer new opportunities to identify mutations in other genes that result in IM and is a necessary first step toward understanding the mechanisms of both tumor growth and regression and its targeted treatment.

TRPV1 Potentiates TGFβ-Induction of Corneal Myofibroblast Development through an Oxidative Stress-Mediated p38-SMAD2 Signaling Loop

Injuring mouse corneas with alkali causes myofibroblast expression leading to tissue opacification. However, in transient receptor potential vanilloid 1 channel (TRPV1-/-) knockout mice healing results in transparency restoration. Since TGFβ is the primary inducer of the myofibroblast phenotype, we examined the mechanism by which TRPV1 affects TGFβ-induced myofibroblast development. Experiments were performed in pig corneas and human corneal fibroblasts (HCFs). Immunohistochemical staining of α-smooth muscle actin (α-SMA) stress fibers was used to visualize myofibroblasts. Protein and phosphoprotein were determined by Western blotting. siRNA transfection silenced TRPV1 gene expression. Flow cytometry with a reactive oxygen species (ROS) reporting dye analyzed intracellular ROS. [Ca2+]I was measured by loading HCF with fura2. In organ cultured corneas, the TRPV1 antagonist capsazepine drastically reduced by 75% wound-induced myofibroblast development. In HCF cell culture, TGF-β1 elicited rapid increases in Ca2+ influx, phosphorylation of SMAD2 and MAPKs (ERK1/2, JNK1/2 and p38), ROS generation and, after 72 hrs myofibroblast development. SMAD2 and p38 activation continued for more than 16 h, whereas p-ERK1/2 and p-JNK1/2 waned within 90 min. The long-lived SMAD2 activation was dependent on activated p38 and vice versa, and it was essential to generate a > 13-fold increase in α-SMA protein and a fully developed myofibroblast phenotype. These later changes were markedly reduced by inhibition of TRPV1 or reduction of the ROS generation rate. Taken together our results indicate that in corneal derived fibroblasts, TGFβ- induced myofibroblast development is highly dependent on a positive feedback loop where p-SMAD2-induced ROS activates TRPV1, TRPV1 causes activation of p38, the latter in turn further enhances the activation of SMAD2 to establish a recurrent loop that greatly extends the residency of the activated state of SMAD2 that drives myofibroblast development.

Syndecan-1 regulates cell migration and fibronectin fibril assembly

Corneal scarring is a major cause of blindness worldwide and can result from the deposition of abnormal amounts of collagen fibers lacking the correct size and spacing required to produce a clear cornea. Collagen fiber formation requires a preformed fibronectin (FN) matrix. We demonstrate that the loss of syndecan1 (sdc1) in corneal stromal cells (CSC) impacts cell migration rates, the sizes and composition of focal and fibrillar adhesions, the activation of integrins, and the assembly of fibronectin into fibrils. Integrin and fibronectin expression are not altered on sdc1-null CSCs. Cell adhesion, spreading, and migration studies using low compared to high concentrations of FN and collagen I (CNI) or vitronectin (VN) with and without activation of integrins by manganese chloride show that the impact of sdc1 depletion on integrin activation varies depending on the integrin-mediated activity evaluated. Differences in FN fibrillogenesis and migration in sdc1-null CSCs are reversed by addition of manganese chloride but cell spreading differences remain. To determine if our findings on sdc1 were specific to the cornea, we compared the phenotypes of sdc1-null dermal fibroblasts with those of CSCs. We found that without sdc1, both cell types migrate faster; however, cell-type-specific differences in FN expression and its assembly into fibrils exist between these two cell types. Together, our data demonstrate that sdc1 functions to regulate integrin activity in multiple cell types. Loss of sdc1-mediated integrin function results in cell-type specific differences in matrix assembly. A better understanding of how different cell types regulate FN fibril formation via syndecans and integrins will lead to better treatments for scarring and fibrosis.

TGF-β–Stimulated CTGF Production Enhanced by Collagen and Associated with Biogenesis of a Novel 31-kDa CTGF Form in Human Corneal Fibroblasts

PURPOSE Connective tissue growth factor (CTGF) is induced by transforming growth factor-beta (TGF-β) after corneal wounding. This study addressed the role of the extracellular matrix in the induction of CTGF by TGF-β. METHODS Human corneal fibroblasts (HCFs) were grown on fibronectin (FN), vitronectin (VN), or collagen (CL) in supplemented serum-free media alone or with TGF-β1 or fibroblast growth factor plus heparin. CTGF mRNA was analyzed by qPCR and protein expression by Western blot analysis of Triton X-100 (TX-100)-soluble and TX-100-insoluble cell lysates using antibodies to N-terminal, mid, and C-terminal CTGF regions. Immunocytochemistry was performed on nonconfluent or scrape-wounded confluent HCFs. RESULTS TGF-β-treated HCFs grown on CL produced five times more 38-kDa CTGF than untreated controls (72 hours). TGF-β-treated HCFs on CL secreted twofold more CTGF than those on FN or VN. Furthermore, a 31-kDa CTGF form, lacking the N-terminal domain, was detected in Triton X-100 insoluble fractions in Western blot analysis. Immunodetectable extracellular CTGF formed linear arrays parallel to, but not colocalized with, CL or FN. It also did not colocalize with FAK, vinculin, or integrins α(v)β(3) and α(5)β(1). Intracellular CTGF was detected in the Golgi apparatus and vesicles, including endosomes. CONCLUSIONS Enhanced CTGF secretion induced by TGF-β in CL-grown cells may contribute to positive feedback in which CL is overexpressed in CTGF-induced fibrosis. N-terminal CTGF fragments in the plasma of patients with severe fibrotic disease may be a product of CTGF proteolysis that also produces the newly identified 31-kDa CTGF that remains cell associated and may have its impact by non-integrin signaling pathways.

Degradation of internalized αvβ5 integrin is controlled by uPAR bound uPA: effect on β1 integrin activity and α-SMA stress fiber assembly.

Myofibroblasts (Mfs) that persist in a healing wound promote extracellular matrix (ECM) accumulation and excessive tissue contraction. Increased levels of integrin αvβ5 promote the Mf phenotype and other fibrotic markers. Previously we reported that maintaining uPA (urokinase plasminogen activator) bound to its cell-surface receptor, uPAR prevented TGFβ-induced Mf differentiation. We now demonstrate that uPA/uPAR controls integrin β5 protein levels and in turn, the Mf phenotype. When cell-surface uPA was increased, integrin β5 levels were reduced (61%). In contrast, when uPA/uPAR was silenced, integrin β5 total and cell-surface levels were increased (2–4 fold). Integrin β5 accumulation resulted from a significant decrease in β5 ubiquitination leading to a decrease in the degradation rate of internalized β5. uPA-silencing also induced α-SMA stress fiber organization in cells that were seeded on collagen, increased cell area (1.7 fold), and increased integrin β1 binding to the collagen matrix, with reduced activation of β1. Elevated cell-surface integrin β5 was necessary for these changes after uPA-silencing since blocking αvβ5 function reversed these effects. Our data support a novel mechanism by which downregulation of uPA/uPAR results in increased integrin αvβ5 cell-surface protein levels that regulate the activity of β1 integrins, promoting characteristics of the persistent Mf.

Expression of Insulin-Like Growth Factor 2 Receptor in Corneal Keratocytes During Differentiation and in Response to Wound Healing

PURPOSE Insulin-like growth factor 2 receptor (IGF2R) associates with ligands that influence wound healing outcomes. However, the expression pattern of IGF2R and its role in the cornea is unknown. METHODS Human keratocytes were isolated from donor corneas. Fibroblasts (fibroblast growth factor 2 [FGF2]-treated) or myofibroblasts (TGF-β1-treated) were analyzed for IGF2R and α-smooth muscle actin (α-SMA) expression by Western blotting and immunolocalization. Mouse corneas were wounded in vivo and porcine corneas ex vivo. The IGF2R and α-SMA protein expression were visualized and quantified by immunohistochemistry. The IGF2R gene expression in human corneal fibroblasts was knocked-down with targeted lentiviral shRNA. RESULTS The IGF2R is expressed in epithelial and stromal cells of normal human, mouse, and porcine corneas. The IGF2R increases (11.2 ± 0.4-fold) in the epithelial and (11.7 ± 0.9-fold) stromal layers of in vivo wounded mouse corneas. Double-staining with α-SMA- and IGF2R-specific antibodies reveals that IGF2R protein expression is increased in stromal myofibroblasts in the wounded cornea relative to keratocytes in the normal cornea (11.2 ± 0.8-fold). Human primary stromal keratocytes incubated with FGF2 or TGF-β1 in vitro demonstrate increased expression (2.0 ± 0.4-fold) of IGF2R in myofibroblasts relative to fibroblasts. Conversion of IGF2R shRNA-lentiviral particle transduced corneal fibroblasts to myofibroblasts reveals a dependence on IGF2R expression, as only 40% ± 10% of cells transduced converted to myofibroblasts compared to 86% ± 3% in control cells. CONCLUSIONS The IGF2R protein expression is increased during corneal wound healing and IGF2R regulates human corneal fibroblast to myofibroblast differentiation.

uPA binding to PAI-1 induces corneal myofibroblast differentiation on vitronectin.

PURPOSE Vitronectin (VN) in provisional extracellular matrix (ECM) promotes cell migration. Fibrotic ECM also includes VN and, paradoxically, strongly adherent myofibroblasts (Mfs). Because fibrotic Mfs secrete elevated amounts of urokinase plasminogen activator (uPA), we tested whether increased extracellular uPA promotes the persistence of Mfs on VN. METHODS Primary human corneal fibroblasts (HCFs) were cultured in supplemented serum-free medium on VN or collagen (CL) with 1 ng/mL transforming growth factor β1 (TGFβ1). Adherent cells were quantified using crystal violet. Protein expression was measured by Western blotting and flow cytometry. Transfection of short interfering RNAs was performed by nucleofection. Mfs were identified by α-smooth muscle actin (α-SMA) stress fibers. Plasminogen activator inhibitor (PAI-1) levels were quantified by ELISA. RESULTS TGFβ1-treated HCFs secreted PAI-1 (0.5 uM) that bound to VN, competing with αvβ3/αvβ5 integrin/VN binding, thus promoting cell detachment from VN. However, addition of uPA to cells on VN increased Mf differentiation (9.7-fold), cell-adhesion (2.2-fold), and binding by the VN integrins αvβ3 and -β5 (2.2-fold). Plasmin activity was not involved in promoting these changes, as treatment with the plasmin inhibitor aprotinin had no effect. A dominant negative PAI-1 mutant (PAI-1R) that binds to VN but does not inhibit uPA prevented the increase in uPA-stimulated cell adhesion and reduced uPA-stimulated integrin αvβ3/αvβ5 binding to VN by 73%. CONCLUSIONS uPA induction of TGFβ1-dependent Mf differentiation on VN supports the hypothesis that elevated secretion of uPA in fibrotic tissue may promote cell adhesion and the persistence of Mfs. By blocking uPA-stimulated cell adhesion, PAI-1R may be a useful agent in combating corneal scarring.

Autophagy and Mitochondrial Dysfunction in Tenon Fibroblasts from Exfoliation Glaucoma Patients

Purpose To test the hypothesis that autophagy dysfunction is involved in exfoliation syndrome (XFS), a systemic disorder of extracellular elastic matrices that causes a distinct form of human glaucoma. Methods Fibroblasts derived from tenon tissue discards (TFs) from filtration surgery to relieve intraocular pressure in XFS patients were compared against age-matched TFs derived from surgery in primary open-angle glaucoma (POAG) patients or from strabismus surgery. Differential interference contrast light, and electron microscopy were used to examine structural cell features. Immunocytochemistry was used to visualize LOXL1 and Fibulin-5, lysosomes, endosomes, Golgi, and microtubules. Light scatter, Cyto-IDTM and JC1 flow cytometry were used to measure relative cell size, autophagic flux rate and mitochondrial membrane potential (MMPT), respectively. Enhanced autophagy was induced by serum withdrawal. Results In culture, XFS-TFs were 1.38-fold larger (by light scatter ratio, p = 0.05), proliferated 42% slower (p = 0.026), and were morphologically distinct in 2D and 3D culture compared to their POAG counterparts. In extended 3D cultures, XFS-TFs accumulated 8–10 times more Fibulin-5 than the POAG-TFs, and upon serum withdrawal, there were marked deficiencies in relocation of endosomes and lysosomes to the perinuclear area. Correspondingly, the XFS-TFs displayed significant accumulation of the autophagasome marker LC3 II (3.9 fold increase compared to POAG levels, p = 0.0001) and autophagic flux rate as measured by Cyto-ID dye was 53% lower in XFS-TFs than in POAG-TFs (p = 0.01), indicating reduced clearance of autophagasomes. Finally the percent of cells with diminished MMPT was 3–8 times larger in the XFS-TFs than in POAG-TFs (p = 0.02). Conclusions Our results provide for the first time a link between XFS pathology to autophagy dysfunction, a major contributor to multiple age related diseases systemically throughout the body, in the brain and in the retina. A diminished capacity for degradation of denatured protein and aging cellular organelles may underpin the development of extracellular protein aggregates in XFS.

The deubiquitylase USP10 regulates integrin β1 and β5 and fibrotic wound healing

ABSTRACT Scarring and fibrotic disease result from the persistence of myofibroblasts characterized by high surface expression of αv integrins and subsequent activation of the transforming growth factor β (TGFβ) proteins; however, the mechanism controlling their surface abundance is unknown. Genetic screening revealed that human primary stromal corneal myofibroblasts overexpress a subset of deubiquitylating enzymes (DUBs), which remove ubiquitin from proteins, preventing degradation. Silencing of the DUB USP10 induces a buildup of ubiquitin on integrins β1 and β5 in cell lysates, whereas recombinant USP10 removes ubiquitin from these integrin subunits. Correspondingly, the loss and gain of USP10 decreases and increases, respectively, αv/β1/β5 protein levels, without altering gene expression. Consequently, endogenous TGFβ is activated and the fibrotic markers alpha-smooth muscle actin (α-SMA) and cellular fibronectin (FN-EDA) are induced. Blocking either TGFβ signaling or cell-surface αv integrins after USP10 overexpression prevents or reduces fibrotic marker expression. Finally, silencing of USP10 in an ex vivo cornea organ culture model prevents the induction of fibrotic markers and promotes regenerative healing. This novel mechanism puts DUB expression at the head of a cascade regulating integrin abundance and suggests USP10 as a novel antifibrotic target. Highlighted Article: The deubiquitylase USP10 regulates αv integrin protein levels, fibrotic markers and myofibroblast persistence in a wound. Ubiquitin-mediated pathways should thus be considered in the pathogenesis of fibrotic healing.