Skip to main content
NCBI home page
American Journal of Physiology - Gastrointestinal and Liver Physiology logoLink to American Journal of Physiology - Gastrointestinal and Liver Physiology
. 2021 Apr 28;320(6):G1044–G1053. doi: 10.1152/ajpgi.00105.2021

Targeting myosin 1c inhibits murine hepatic fibrogenesis

Ehtesham Arif 1,2, Cindy Wang 2, Marzena K Swiderska-Syn 3, Ashish K Solanki 1, Bushra Rahman 1, Paul P Manka 2,4, Jason D Coombes 5,6, Ali Canbay 4, Salvatore Papa 7, Deepak Nihalani 1,8, Patricia Aspichueta 9, Joshua H Lipschutz 1,10, Wing-Kin Syn 2,9,11,✉
PMCID: PMC8285590  PMID: 33908271

graphic file with name gi-00105-2021r01.jpg

Open in a new tab

Keywords: hepatic fibrosis, Myo1c, TGF-β signaling

Abstract

Myosin 1c (Myo1c) is an unconventional myosin that modulates signaling pathways involved in tissue injury and repair. In this study, we observed that Myo1c expression is significantly upregulated in human chronic liver disease such as nonalcoholic steatohepatitis (NASH) and in animal models of liver fibrosis. High throughput data from the GEO-database identified similar Myo1c upregulation in mice and human liver fibrosis. Notably, transforming growth factor-β1 (TGF-β1) stimulation to hepatic stellate cells (HSCs), the liver pericyte and key cell type responsible for the deposition of extracellular matrix, upregulates Myo1c expression, whereas genetic depletion or pharmacological inhibition of Myo1c blunted TGF-β-induced fibrogenic responses, resulting in repression of α-smooth muscle actin (α-SMA) and collagen type I α 1 chain (Col1α1) mRNA. Myo1c deletion also decreased fibrogenic processes such as cell proliferation, wound healing response, and contractility when compared with vehicle-treated HSCs. Importantly, phosphorylation of mothers against decapentaplegic homolog 2 (SMAD2) and mothers against decapentaplegic homolog 3 (SMAD3) were significantly blunted upon Myo1c inhibition in GRX cells as well as Myo1c knockout (Myo1c-KO) mouse embryonic fibroblasts (MEFs) upon TGF-β stimulation. Using the genetic Myo1c-KO mice, we confirmed that Myo1c is critical for fibrogenesis, as Myo1c-KO mice were resistant to carbon tetrachloride (CCl4)-induced liver fibrosis. Histological and immunostaining analysis of liver sections showed that deposition of collagen fibers and α-SMA expression were significantly reduced in Myo1c-KO mice upon liver injury. Collectively, these results demonstrate that Myo1c mediates hepatic fibrogenesis by modulating TGF-β signaling and suggest that inhibiting this process may have clinical application in treating liver fibrosis.

NEW & NOTEWORTHY The incidences of liver fibrosis are growing at a rapid pace and have become one of the leading causes of end-stage liver disease. Although TGF-β1 is known to play a prominent role in transforming cells to produce excessive extracellular matrix that lead to hepatic fibrosis, the therapies targeting TGF-β1 have achieved very limited clinical impact. This study highlights motor protein myosin-1c-mediated mechanisms that serve as novel regulators of TGF-β1 signaling and fibrosis.

INTRODUCTION

Chronic liver injury triggers a repair response that leads to liver fibrosis (i.e., liver scarring), which is characterized by the excessive accumulation of collagenous extracellular matrices (ECMs). This repair response occurs as a result of various insults including toxins, autoimmune disorders, cholestatic or metabolic diseases or viral infections, and leads to the development of fibrosis, cirrhosis, and cirrhosis-associated complications such as portal hypertension, hepatocellular carcinoma, and liver failure (1–3). Hepatic stellate cells (HSCs) are the liver pericytes that are considered the principal source of extracellular matrix proteins including collagen in the liver. Complex molecular mechanisms and signaling pathways play critical roles in the fibrogenic process, and transforming growth factor-β1 (TGF-β1) is a prototypical profibrogenic cytokine that activates HSC and promotes collagen deposition (4). Intracellular signal transducers Smads whose phosphorylation and subsequent translocation into the nucleus upon TGF-β1 activation regulate expression of profibrotic target genes that contributes to collagen synthesis and liver fibrosis (5, 6).

Myo1c is an unconventional class I myosin and an actin-based molecular motor, which is actively involved in various cellular functions including intracellular trafficking, cell adhesion, motility, and maintenance of membrane tension (7–10). Myo1c is expressed in various cell types and is generally associated with actin-rich cortical membrane structures such as filopodia, lamellipodia, and ruffles (11). The Myo1c gene has two isoforms, which are referred to as cytoplasmic (cMYO1C) and nuclear Myo1c (NM1) (12). NM1 differs from cMYO1C by the presence of 16 or 35 additional amino acids at the NH2-terminus. Although the cMYO1C interacts with various proteins and participates in cellular functions, NM1 is involved in chromatin remodeling, transcription, mRNA maturation, and chromosome movement (12–14). Our previous study showed that NM1 targets TGF-β responsive gene GDF-15 that contributes to the fibrogenic process in podocytes (10). Various cellular and signaling functions were also observed that confirmed the role of cMYO1C in fibrosis. Notably, another study showed that the Myo1c inhibitor pentachloropseudilin (PCIP) inhibited TGF-β activity by accelerating TGF-β receptor turnover. PCIP attenuated TGF-β-induced smad2/3 phosphorylation and repressed expression of vimentin, N-cadherin, and fibronectin and, thus, blocking TGF-β-induced epithelial to mesenchymal transition (EMT) (7). Since TGF-β-signaling plays a major role in hepatic fibrogenesis, we evaluated the pathophysiological significance of Myo1c in hepatic fibrogenesis using in vitro and in vivo models.

METHODS

Cell Culture

The clonally derived rat myofibroblastic hepatic stellate cell (HSCs/GRX) (15) were cultured in DMEM media supplemented with 10% FBS and 1% pen/strep as described earlier (16). Mouse embryonic fibroblasts (MEFs) were isolated from Myo1c-flox/flox mice using embryos at E13.5 from the pregnant females as described earlier (17). MEFs were also cultured in DMEM medial and supplemented with 10% FBS, 1% penicillin/streptomycin, and 1% nonessential amino acids. All these cells were plated on a 10 cm2 dish and incubated at 37°C in the presence of 5% CO2. The Myo1c inhibitor PClP was used at a concentration of 1 μM (18). These cells were stimulated with TGF-β as described (10). Briefly, cells were incubated overnight in DEME medium with 0.1% FBS and stimulated with 5 ng/mL of TGF-β in the same medium for a period of 48 hours.

Generation of Myo1c-Knockdown and Knockout Cells

Myo1c knockdown in GRX cells were generated using lentiviral vector carrying Myo1c shRNA (TRCN0000100742, Sigma) and control knockdown cells were generated using scramble lentiviral vector (SHC016V, Sigma). MEFs Myo1c-KO and control-KO cells were generated by transducing adenovirus either adeno-cre or adeno-β-gal virus for 24 h and experiments were performed after 72–96 h of posttransduction.

Quantitative Reverse-Transcription Polymerase Chain Reaction

Total RNA was isolated from cultured cell using TRIzol method (Life Technologies, Carlsbad, CA) as per manufacturer’s guidelines with some modification as described earlier (19). First-strand cDNA was synthesized using the iScript Select cDNA Synthesis Kit and 1.0 μg of total RNA was used (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer’s instructions. Quantitative real-time PCR was performed with iQ SYBR Green super mix, using the iCycler iQ Real time PCR Detection System (Bio-Rad). Details of the primers used for Myo1c, α-SMA, and Col1α1 are described earlier (10, 20). Mouse ribosomal protein S9 (RPS9) primer was used as a control to normalize the expression (20).

Western Blot Analysis

Cultured cells were lysed in RIPA buffer and protein estimation was performed using the bicinchoninic acid (BCA) method. Total protein samples of 20 μg from lysates were used for Western blot analysis as described earlier (10). Mouse monoclonal Myo1c antibody was used as reported earlier (10), whereas other antibodies, including GAPDH (Sigma no. G8795), α-SMA (Santa Cruz no. c-53142), and Actin (Santa Cruz no. sc-47778) were commercially obtained. Western blotting image acquisition and densitometric analysis was performed using LI-CORE imaging station as described earlier.

Cell Migration Assay

Cell migration assay was performed as described earlier with some modification (21). Briefly, control and Myo1c knockdown GRX cells were grown in 35-mm glass-bottom culture dishes (Mat-Tek Corporation) until they reached confluence. Scratches were created using a 200-μL sterile pipette tip and images were taken at 0 h, 8 h, 16 h, and 24 h. ImageJ (National Institutes of Health) was used to calculate the length of the wound's closure. The experiment was performed more than three times, and the distance (μM) of migration was calculated.

Cell Proliferation Assay

Cell proliferation was determined using Cell Counting Kit-8 (CCK-8) as per manufacturer's instruction. Briefly, cells were plated in 96-well plates and incubated with CCK-8 solution for different time points such as 17 h, 41 h, and 65 h for GRX cells. Absorbance at 450 nm was measured using a microplate reader and extent of proliferation was measured. Cell proliferation was also determined using SRB Assay/Sulforhodamine B Assay Kit as per manufacturer's instruction. Briefly, in the assay, cultured GRX cells were fixed on plates and stained with Sulforhodamine B. Furthermore, cells were washed and dried, then the bound dye was solubilized and the absorbance at 565 nm was measured.

Gel Contraction Assay

Contractility of control and Myo1c-KD GRX cells were evaluated using collagen gel lattices (PureCol, Advanced Biomatrix) as described earlier (22). Cells were cultured in 24-well culture plates in collagen gel lattice, where it was serum starved and after the dislodgement of the lattice, cells were incubated in DMEM with/without TGF-β1 (5 ng/mL) for 24 h. Collagen gel lattice size was determined at 0 h as well as 24 h post TGF-β treatment and area of gel contraction were calculated.

Treatments of Animals

All experiments were performed in 8- to 12-wk-old mice. A detailed experimental plan for animal treatment is given in Fig. 5C (23, 24). Wild-type and Myo1c-KO mice were treated with CCl4 (0.7 µL/g body wt) or Vehicle (corn oil) through the intraperitoneal injection. Mice were randomly divided into four groups (n = 5 for each group): 1) wild-type control group; treated with vehicle; 2) wild-type experimental group, treated with CCl4; 3) Myo1c-KO control group, treated with vehicles; and 4) Myo1c-KO experimental group, treated with CCl4. At fifth week of the treatment tissues were harvested for the experimental purpose.

Histology and Immunohistochemistry

Mice liver were perfused and washed with ×1 PBS and transected, fixed for 4 to 12 h in 4% paraformaldehyde, rinsed, and sequential alcohol treatments were performed and submitted to the Histology Core facility at Medical University of South Carolina (MUSC) for embedding and sectioning. The paraffin embedded sections were deparaffinized and stained with Masson's trichrome and Sirius Red for histological analysis. Immunohistochemistry was performed as described earlier (10). Briefly, liver sections were deparaffinized and incubated with Tris-EDTA (pH 9.0) buffer for antigen retrieval at 65°C overnight. The sections then blocked with 5% BSA for 1 h at room temperature. Primary antibodies for α-SMA (1:50 dilution) were diluted in 10% goat serum and incubated overnight at 4°C. The sections were washed with ×1 TBS five times and then incubated with Alexa Fluor-labeled secondary antibodies at a dilution of 1:500 for 1 h at 37°C. After washings with TBS these sections were mounted with DAPI and left overnight in the dark for drying and images were collected using fluorescence microscope. Horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies were used for detection for nonfluorescence staining. All parameters were maintained constant throughout the image acquisition, including the exposure time. The image J software was used for quantitative analysis.

Human and Animal Study Approval

Deidentified, formalin-fixed paraffin embedded liver tissue sections (n = 5) from Dr. A. Canbay (09-4252 & 12-5232-BO, Ethics Commission, Medical Faculty of the University of Duisburg-Essen) were stained using established immunohistochemistry protocols. All animal studies were conducted as per the protocol approved by the Medical University of South Carolina, and University of Essen, Institutional Animal Care and Use Committee and NIH Guidelines for the Care and Use of Laboratory Animals (Protocol no. IACUC-2018-00360 of MUSC and Protocol no. G-1593-16 of University of Essen). Treatment of mice, including housing, injections and surgery was in accordance with the institutional guidelines. Isoflurane anesthesia (5% induction, 2% maintenance) was used to perform all surgeries.

RESULTS

Myo1c Expression Was Significantly Increased with Hepatic Fibrosis

The first evidence for the involvement of Myo1c in hepatic fibrosis came from high throughput data. We first examined the gene expression omnibus (GEO) database (GEO no. GDS3087) (25), where microarray and next-generation analysis were performed in livers of animals lacking Trim24 (a ligand-dependent nuclear receptor transcriptional coregulator) and control liver tissue, where the loss of Trim24 was associated with the development of liver fibrosis and HCC (25, 26). We found that Myo1c expression was significantly upregulated in liver tissues with fibrosis and HCC when compared with the normal liver (Fig. 1A). In another throughput data (GEO no. GSE49541) (27), which included patients with a full spectrum of nonalcoholic fatty liver disease (NAFLD) severity, Myo1c expression was significantly higher among those with advanced NAFLD (fibrosis stages 3 and 4), compared with early NAFLD (fibrosis with stages 0 and 1; Fig. 1B).

Figure 1.

Figure 1.

Open in a new tab

Myo1c upregulated during fibrotic injury. A: the GEO profile GDS3087 of Myo1c in hepatocellular tumors of Trim24-deficient mice was retrieved from GEO database and expression pattern of Myo1c was analyzed in normal liver vs hepatocellular tumors. Both, the expression level and rank of Myo1c were significantly increased in hepatocellular tumor as compared with normal liver. B: GEO high throughput data (GEO no. GSE49541), where patients with mild NAFLD (with fibrosis stage 0 and 1) and advanced NAFLD (with fibrosis stage 3 and 4) were analyzed. We retrieve the data and Myo1c expression patterns were analyzed. Expression of Myo1c is found to be significantly upregulated in advanced NAFLD as compared with mild NAFLD. C and D: representative images of liver sections from NASH and normal subjects showed increased Myo1c expression in NASH. Images magnification, ×10 (top) and ×40 (bottom). Quantitative analysis of IHC images showed that Myo1c expression was elevated in NASH as compared with normal liver. P ≤ 0.0001 NASH vs. Normal. Data presented in mean ± SD. E and F: representative images of liver sections from MCD-diet and normal chow diet showed increased Myo1c expression in MCD-diet mice. Images magnification, ×10 (top) and ×40 (bottom). Quantitative analysis of IHC images showed that Myo1c expression was elevated in MCD-diet liver as compared with normal chow-diet liver. P ≤ 0.0001 MCD-diet vs. chow-diet. Data presented in means ± SD. G and H: representative images of liver sections from CCl4-induced mice model and control vehicle treated mice showed increased Myo1c expression in CCl4-induced mice. Images magnification, ×10 (top) and ×40 (bottom). Quantitative analysis of IHC images showed that Myo1c expression was elevated in CCl4-induced liver as compared with normal vehicle-treated liver section. P ≤ 0.0001 MCD-diet vs. Chow-diet. Data presented in means ± SD. I and J: the hepatic stellate cells line (GRX) and mouse embryonic fibroblasts (MEFs) were treated with TGF-β for 48 h and expression of Myo1c was assessed by qPCR and normalized to ribosomal protein S9. Quantitative analysis showed the increased expression of Myo1c in both the cell lines upon TGF-β treatment. Data are from three independent experiments presented as mean ± SD. TGF-β (-) vs. TGF-β (+); P ≤ 0.01. CCl4, carbon tetrachloride; GEO, gene expression omnibus; MCD, methionine-choline deficient; MEFs, mouse embryonic fibroblasts; Myo1c, myosin 1c; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; TGF-β1, transforming growth factor-β1; HCC, hepatocellular carcinoma; IHC, Immunohistochemistry.

Furthermore, Myo1c expression was analyzed through immunohistochemistry in representative liver tissues from humans and animal models of liver fibrosis. Myo1c expression was found to be significantly increased in human non-alcoholic steatohepatitis (NASH) fibrosis when compared with the healthy liver (Fig. 1, C and D). In mice fed the methionine-choline deficient (MCD) diet (a well characterized model of NASH fibrosis) (28) and mice treated with carbon tetrachloride (CCl4) (another model of liver fibrosis) (23), Myo1c expression were also significantly upregulated, when compared with normal chow-fed or vehicle-treated animals, respectively (Fig. 1, E–H). Increased Myo1c expression in CCl4-treated mice were further confirmed by Western blot (Supplemental Fig. S1, A and B; all Supplemental material is available at https://doi.org/10.6084/m9.figshare.14292371.v1). Prolonged feeding with the high-fat-diet (HFD) also induces hepatic fibrogenesis (29); thus, we also examined the expression of Myo1c in these tissues. We found that Myo1c mRNA was significantly increased in livers from mice fed the HFD, which mirrored α-SMA expression (Supplemental Fig. S1, C and D).

Since TGF-β signaling is a prototypical cytokine involved in tissue fibrogenesis, we next evaluated if TGF-β modulated expression of Myo1c. Treatment of the mouse hepatic stellate cells (HSC) line, GRX (15) significantly increased the Myo1c expression (Fig. 1, I and J). We also evaluated responses of mouse embryonic fibroblasts (MEFs) to TGF-β stimulation. Although MEFs are not tissue pericytes, they resemble fibroblasts in culture (17, 30) and were used as another model. We noted similar increases in Myo1c mRNA when mouse MEFs were treated with TGF-β (Fig. 1, I and J).

Targeting Myo1c Attenuated the Fibrogenic Response

Since TGF-β signaling is the primary driver of fibrogenesis (31) and we have shown that Myo1c is upregulated with liver fibrosis, we next evaluated if loss of Myo1c directly inhibits fibrogenesis. To this end, we infected GRX with Myo1c-specific short hairpin RNA (shMyo1c-GRX; which knockdown Myo1c gene expression); we observed >90% repression of Myo1c mRNA (Fig. 2, A and B). Treatment with TGF-β significantly upregulates fibrogenic gene expression (α-SMA and Col1α1) in normal GRX cells, but these changes were significantly blunted in shMyo1c-GRX cells when compared with control (shScr-GRX; i.e., GRX cells infected with scrambled short hairpin RNA; Fig. 2, C and D). To confirm these findings, we repeated experiments using a pharmacological approach. Pentachloropseudilin (PCIP) is a known Myo1c inhibitor, and the treatment of GRX cells with PCIP similarly, led to an attenuated fibrogenic response to TGF-β treatment (Fig. 2, E and F).

Figure 2.

Figure 2.

Open in a new tab

Myo1c deletion attenuates fibrogenic response. A and B: Myo1c knockdown in GRX cell was induced by the lentiviral transfection of Myo1c shRNA, whereas scramble (SCR) transfection was done for controls. Stable transfection was achieved through the puromycin selection and the extent of the Myo1c protein knockdown was assessed by Western blotting (A), whereas qPCR (B) was performed to check knockdown at mRNA level. C and D: to test the TGF-β-induced activation, control and Myo1c knockdown GRX cells were investigated for the expression of the fibrogenic genes. TGF-β significantly upregulates the α-SMA (C) and Col1α1 (D) in control cells, whereas upregulation of these genes was blunted in Myo1c knockdown cells. E and F: effect of Myo1c inhibitor PCIP (0.1 mM) on TGF-β-induced fibrogenic response was analyzed through the qPCR. PCIP treatment significantly blunted the α-SMA expression upon TGF-β, whereas significant differences were observed in Col1α1 expression. Myo1c also contributes dysregulation of cell proliferation, abnormal wound healing, and collagen gel contraction. Cell Counting Kit-8 (CCK-8) was used to measure cell proliferation in control and Myo1c knockdown GRX cells. G: absorbance at 450 nm, which a readout of the number of viable cells is significantly reduced upon Myo1c knockdown at 41 and 65 h. H: analysis of SRB-based cell proliferation further confirmed that there is a significant reduction in cell proliferation in Myo1c knockdown cells. Data are from three independent experiments. I and J: The collagen gel contraction (CGC) assay was performed to analyze the fibrogenic response in presence of TGF-β stimulation in GRX cells. K and L: TGF-β stimulation induced 54% of gel contraction in shScr-GRX (control) cells compared with 17% in shMyo1c-GRX. Myo1c, myosin 1c; n.s., not significant; PCIP, pentachloropseudilin; shMyo1c, Myo1c-specific short hairpin RNA; TGF, transforming growth factor; α-SMA, α-smooth muscle actin; Col1α1, collagen type I α1 chain; OD, optical density. **P < 0.01.

Dysregulation in cell proliferation and migration are associated with fibrogenesis (32, 33), and Myo1c has been reported to play key roles in cell motility, trafficking, cell migration and/or differentiation (8, 11, 34). shScr-GRX (control) and shMyo1c-GRX were analyzed for cell proliferation using the cell counting kit-8 (CCK-8) assay. We found that loss of Myo1c was associated with significantly reduced number of viable cells in shMyo1c-GRX (Fig. 2G). Sulforhodamine B (SRB)-based cell proliferation assay further confirmed the significant reduction in cell proliferation upon Myo1c knockdown (Fig. 2H). Abnormal wound healing results in tissue fibrosis (35, 36), and the scratch assay is widely used to evaluate the wound healing response (37). We found that wound healing was significantly inhibited in shMyo1c-GRX cells compared with control shScr-GRX (Fig. 2, I and J). Semiquantitative analysis confirmed a significant reduction in cell migration distance (in µM) in shMyo1c-GRX cells: shScr-GRX (0.418 µM) versus shMyo1c-GRX (0.254 µM). The collagen gel contraction (CGC) assay is another established method to study the fibrogenic response in presence of various stimuli including TGF-β (38, 39). It also provides information about the collagen remodeling in these cells (39). The effects of Myo1c loss on collagen gel contraction was performed in GRX cells either in presence of TGF-β or vehicle. TGF-β stimulation induced 54% of gel contraction in shScr-GRX (control) cells compared with 17% in shMyo1c-GRX (Fig. 2, K and L). Semiquantitative analysis confirmed a significant reduction in TGF-β1 induced cell contraction upon Myo1c deletion.

To further validate our findings, we generated MEFs from 13.5 day embryos of Myo1c flox/flox mice. We treated these MEFs with either β-gal or Cre virus to generate control and Myo1c knockout MEFs, respectively. Although MEFs are not tissue pericytes, they resemble fibroblasts in culture (17, 30). We observed a significant loss of Myo1c protein in MEFs treated with the cre virus (Fig. 3A). Control (Myo1cfl/fl-MEFs + βGal) and Myo1c-KO (Myo1cfl/fl-MEFs + Cre) MEFs were then treated with TGF-β and fibrogenic response analyzed through qPCR and immunofluorescence. qPCR analysis showed a significant upregulation of α-SMA and Col1α1 expression in control MEFs, but this was attenuated in Myo1c-KO MEFs (Fig. 3, B and C), consistent with changes observed in shMyo1c-GRX cells (Fig. 2, C and D). Immunofluorescence imaging followed by quantitative analysis corroborated our findings, where significant increases in α-SMA protein was detected in control MEFs, but not in Myo1c-KO MEFs (Fig. 3, D and E).

Figure 3.

Figure 3.

Open in a new tab

A: MEFs were isolated from Myo1c flox/flox mice and treated with either β-gal or Cre virus to generate control and Myo1c knockout MEFs, respectively. Western blotting analysis showed that Myo1c proteins were completely knockout after 96 h of postviral treatment. B and C: control (Myo1cfl/fl-MEFs + βGal) and Myo1c-KO (Myo1cfl/fl-MEFs+Cre) MEFs were treated with TGF-β expression of α-SMA and Col1α1 genes were analyzed, where significant upregulation of these genes were observed in control MEFs but attenuated in Myo1c-KO MEFs. D: TGF-β-induced expression of α-SMA was analyzed through immunofluorescence staining, where cells MEFs were stained with α-SMA (green) antibody and mounted with DAPI (blue). E: quantitative analysis of fluorescence means pixel intensity showed increased expression of α-SMA in control MEFs as compared with Myo1c-KO MEFs (n = 50 cells). Data are from three independent experiments presented as means ± SD. N.S: nonsignificant. P ≤ 0.05, Significant. MEFs, mouse embryonic fibroblasts; Myo1c, myosin 1c; TGF, transforming growth factor; α-SMA, α-smooth muscle actin; Col1α1, collagen type I α1 chain.

Loss of Myo1c Attenuated TGF-β Signaling

We next evaluated if attenuation of fibrogenic responses observed with Myo1c inhibition (pharmacologic) and Myo1c deletion (genetic) were associated with the changes in downstream components of the TGF-β signaling pathway. GRX cells were treated with TGF-β or vehicles in presence or absence of PCIP, a pharmacological inhibitor of Myo1c (18). Western blotting analysis showed significant reduction in phosphorylation of SMAD2 and SMAD3 upon Myo1c inhibition in GRX cells (Fig. 4, A and B). Control or Myo1c-KO MEFs were similarly treated with TGF-β for 48 h; at the end of treatment, cells were harvested, and Western blot performed. We detected a comparable reduction in levels of phosphorylated SMAD2 and SMAD3 in Myo1c-KO MEFs (Fig. 4, C and D). These results in aggregate, confirm that loss Myo1c inhibits liver fibrogenesis.

Figure 4.

Figure 4.

Open in a new tab

TGF-β-induced signaling is blunted upon Myo1c inhibition and in Myo1c-deletion. A and B: the signaling components of TGF-β signaling pathways in vehicle and PCIP-treated GRX cells were screened using Western blotting. Quantitative analysis showed reduced phosphorylation of SMAD2 and SMAD3 expression in Myo1c-KO MEFs. Data are presented in mean ± SD. C and D: similarly, the signaling components of TGF-β signaling pathways in control and Myo1c-KO MEFs were screened using Western blotting. Quantitative analysis showed reduced phosphorylation of SMAD2 and SMAD3 expression in Myo1c-KO MEFs. Data are presented in means ± SD. KO, knockout; MEFs, mouse embryonic fibroblasts; Myo1c, myosin 1c; PCIP, pentachloropseudilin; TGF, transforming growth factor; SMAD, mothers against decapentaplegic homolog.

Myo1c Deletion Attenuated CCl4-Induced Liver Fibrosis

To understand the physiological significance of Myo1c, we used global Myo1c knockout mice as described in our previous work (10). In brief, Myo1c flox/flox mice were crossed with CMV cre mice obtained from the Jackson laboratory [B6.C-Tg(CMV-cre)1Cgn/J; Stock No: 006054]. Cre recombination deletes the critical 5–13 exon of Myo1c as presented in Fig. 5A. Deletion of Myo1c was confirmed through the Western blotting analysis using specific Myo1c antibody (Fig. 5B). Wild-type and Myo1c-KO mice were treated with either CCl4 (0.7 μL/g body weight) or Vehicle (corn oil) and tissue were harvested at 5th week (23, 24, 40). Schematic figure of experimental design is presented in Fig. 5C. At the end of study, livers were harvested and stained with Masson’s trichrome and Sirius-red to assess the degree of fibrosis (Fig. 5, D and F). Loss of Myo1c resulted in significantly less liver fibrosis compared with control mice (Fig. 5D). Semiquantitative analysis of Masson’s trichrome images showed 8.2% of area with collagen deposition in control versus 3.37% in Myo1c-KO mice (P < 0.001; Fig. 5E). Similar changes were seen with Sirius-red staining: 9.7% of area with collagen deposition in control versus 5.7% in Myo1c-KO mice (Fig. 5G).

Figure 5.

Figure 5.

Open in a new tab

Myo1c deletion attenuates CCl4-induced liver fibrosis in mice. A: global Myo1c mice were generated using Myo1c flox/flox mice as presented in schematic figure. Myo1c flox/flox mice were crossed with CMV cre (B6.C-Tg(CMV-cre)1Cgn/J; Stock No: 006054), which eventually leads to deletion of the critical 5–13 exon of the Myo1c. B: deletion of Myo1c was confirmed through the Western blotting analysis using specific Myo1c antibody, which showed complete knockout of Myo1c in liver and kidney. C: schematic figure showing experimental design including timelines of CCl4 treatment and end of the study. D: increased Masson’s trichrome staining was noted in the liver of wild-type mice as compared with Myo1c-KO mice treated with CCl4. Scale bars: 50 μm. E: fibrotic area assessment from the Masson’s trichrome stained liver of wild-type mice showed ∼8.2% fibrosis, whereas the Myo1c-KO showed ∼3.37% fibrosis. P ≤ 0.001, wild-type-CCl4 vs. Myo1c-KO-CCl4, n = 5 mice in each group using manual outlining method. Data presented in mean ± SD. F: increased Sirius-red staining was also noted in the liver of wild-type mice as compared with Myo1c-KO mice treated with CCl4. Scale bars: 50 μm. G: fibrotic area assessment from the Sirius-red stained liver of wild-type mice showed ∼9.7% fibrosis, whereas the Myo1c-KO showed ∼5.7% fibrosis. P ≤ 0.001, wild-type-CCl4 vs. Myo1c-KO-CCl4, n = 5 mice in each group using manual outlining method. Data presented in means ± SD. CCl4, carbon tetrachloride; KO, knockout; Myo1c, myosin 1c.

α-SMA is an actin isoform and a specific marker for HSC activation and fibrogenesis (41). Loss of Myo1c was associated with significantly fewer α-SMA positive cells by immunofluorescence and immunohistochemistry (Fig. 6, A–C). Analysis of mean pixel intensity in fluorescence images and α-SMA-positive area also confirmed the significant increase of α-SMA protein expression in CCl4-treated wild-type mice but this was significantly repressed in Myo1c-KO mice (Fig. 6, B and D). Collectively, these results showed that loss of Myo1c attenuated CCl4-induced liver fibrosis in mice.

Figure 6.

Figure 6.

Open in a new tab

A: immunostaining of liver sections using α-SMA antibody and DAPI (blue) showed increased α-SMA expression in wild-type mice in response to CCl4-induced injury. Scale bars: 20 μm. B: quantitative analysis of immunofluorescence images showed that CCl4 injury-induced α-SMA expression was elevated in wild-type mice when compared with Myo1c-KO mice. P ≤ 0.01 wild-type (CCl4) vs. Myo1c-KO (CCl4). n = 5 mice in each group. Data presented as means ± SD. C: immunostaining of liver sections using α-SMA antibody and HRP conjugated secondary antibody showed increased α-SMA positive area in wild-type mice in response to CCl4-induced injury. (top ×10; bottom ×40 magnification). D: quantitative analysis of α-SMA-positive area showed that CCl4 injury induced significant increase in α-SMA-positive area in wild-type mice when compared with Myo1c-KO mice. Data presented in means ± SD. CCl4, carbon tetrachloride; KO, knockout; Myo1c, myosin 1c; α-SMA, α-smooth muscle actin; ZO1, Zonula occludens-1.

DISCUSSION

The mechanisms of tissue fibrogenesis is complex and TGF-β1 dependent signaling is a prototypical pathway involved in liver fibrosis. In this study, we showed that the unconventional class I myosin and actin-based molecular motor is a key modulator of liver fibrosis. In a series of in vitro and in vivo studies, we showed that Myo1c is upregulated in liver fibrosis, where it enhanced canonical TGF-β signaling. Conversely, targeting Myo1c pharmacologically or genetically significantly reduced levels of phosphorylated Smads and alleviated the liver repair response.

This novel role of Myo1c in tissue fibrogenesis simply recapitulates its function in cell adhesion, motility and maintenance of membrane tension (8, 9, 11). Interestingly, Myo1c is also highly expressed in adipocytes where it facilitates recycling of the glucose transporter (42); specifically, by regulating the trafficking of intracellular GLUT4-containing vesicles to the plasma membrane in response to insulin (42). This is relevant to fibrogenesis because recent data show that metabolic reprogramming can regulate HSC activation and fibrogenic responses (43). Myo1c also stabilizes actin and participates as an important mediator of VEGF-induced VEGFR2 delivery to the cell surface and plays a role in angiogenic signaling (44), a feature characteristic of liver fibrosis. These studies in aggregate, support the role for Myo1c in hepatic fibrogenesis, and is consistent with our previous study where we had shown that Myo1c regulates fibrogenesis in kidney podocytes (10), and another group had reported that treatment with PCIP repressed TGF-β-induced epithelial to mesenchymal transition (EMT) (7).

Although Myo1c appears to be a critical modulator of canonical TGF-β signaling, the mechanisms by which Myo1c effects changes in the levels of Smad phosphorylation is unclear but is likely to involve both isoforms of the Myo1c protein. Studies to date suggest that the cytoplasmic isoform is involved in intracellular trafficking and the stabilization of key cellular proteins, whereas the nuclear isoform regulates chromatin remodeling and gene expression (8, 12, 14, 34, 42, 45). It is therefore, possible that Myo1c could regulate adaptor proteins or surface expression of TGF-β receptors (similar to its regulation of VEGFR2 expression). As growth differentiation factor (GDF)15 is a downstream effector of Myo1c (10) and has recently been shown to directly activate lung fibroblasts and macrophages (46), future studies will be needed to determine if Myo1c-associated GDF15 secretion also contributes to the profibrogenic milieu in chronic liver disease. These latter studies are important because Myo1c is ubiquitously expressed and the generalized targeting of Myo1c would likely lead to adverse clinical outcomes; targeting downstream effectors such as GDF15, and/or in a tissue-/cell-selective manner would significantly mitigate side effects.

In conclusion, we showed for the first time that Myo1c plays an important role in regulating hepatic fibrogenesis and that targeting Myo1c protects mice from liver fibrosis.

SUPPLEMENTAL MATERIAL

Supplemental material is available at https://doi.org/10.6084/m9.figshare.14292371.v1.

GRANTS

This study is supported by the Division of Gastroenterology and Hepatology, Medical University of South Carolina (W-K.S), the Ralph H Johnson Veterans Affairs Medical Center (W-K.S), and National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, Grant RO3 5R03TR003038-02 (E.A.), and Rosetrees Trust, Seedcorn Grant M894 (S.P.) and Guts UK, Development Grant DGO2019_02 (S.P.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

E.A., D.N., and W-K.S. conceived and designed research; E.A., C.W., M.K.S., A.K.S., S.P., B.R., and P.P.M. performed experiments; E.A., C.W., and P.P.M. analyzed data; E.A., D.N., W-K.S., and J.D.C. interpreted results of experiments; E.A. and C.W. prepared figures; E.A. and W-K.S. drafted manuscript; E.A., J.H.L., W-K.S., J.D.C., A.C., and S.P. edited and revised manuscript; E.A. and W-K.S. approved final version of manuscript.

REFERENCES

  • 1.Yi H-S, Eun HS, Lee Y-S, Jung JY, Park S-H, Park K-G, Choi S-K, Suh J-M, Jeong W-I-G. Treatment with 4-methylpyrazole modulated stellate cells and natural killer cells and ameliorated liver fibrosis in mice. PLoS One 10: e0127946, 2015. doi: 10.1371/journal.pone.0127946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Tanaka M, Miyajima A. Liver regeneration and fibrosis after inflammation. Inflamm Regen 36: 19, 2016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Bataller R, Brenner DA. Liver fibrosis. J Clin Invest 115: 209–218, 2005. doi: 10.1172/JCI24282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Eghbali-Fatourechi G, Sieck GC, Prakash YS, Maercklein P, Gores GJ, Fitzpatrick LA. Type I procollagen production and cell proliferation is mediated by transforming growth factor-beta in a model of hepatic fibrosis. Endocrinology 137: 1894–1903, 1996. doi: 10.1210/endo.137.5.8612529. [DOI] [PubMed] [Google Scholar]
  • 5.Gressner AM, Weiskirchen R. Modern pathogenetic concepts of liver fibrosis suggest stellate cells and TGF-beta as major players and therapeutic targets. J Cellular Mol Med 10: 76–99, 2006. doi: 10.1111/j.1582-4934.2006.tb00292.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Lu N, Liu Y, Tang A, Chen L, Miao D, Yuan X. Hepatocyte-specific ablation of PP2A catalytic subunit alpha attenuates liver fibrosis progression via TGF-beta1/Smad signaling. Biomed Res Int 2015: 794862, 2015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Chung C-L, Wang S-W, Martin R, Knölker H-J, Kao Y-C, Lin M-H, Chen J-J, Huang Y-B, Wu D-C, Chen C-L. Pentachloropseudilin inhibits transforming growth factor-beta (TGF-beta) activity by accelerating cell-surface type II TGF-beta receptor turnover in target cells. Chembiochem 19: 851–864, 2018. doi: 10.1002/cbic.201700693. [DOI] [PubMed] [Google Scholar]
  • 8.Capmany A, Yoshimura A, Kerdous R, Caorsi V, Lescure A, Nery E-D, Courdrier E, Goud B, Schauer K. MYO1C stabilizes actin and facilitates the arrival of transport carriers at the Golgi complex. J Cell Sci 132: jcs225029, 2019. [DOI] [PubMed] [Google Scholar]
  • 9.Venit T, Kalendová A, Petr M, Dzijak R, Pastorek L, Rohožková J, Malohlava J, Hozak P. Nuclear myosin I regulates cell membrane tension. Sci Rep 6: 30864, 2016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Arif E, Solanki AK, Srivastava P, Rahman B, Tash BR, Holzman LB, Janech MG, Martin R, Knolker HJ, Fitzgibbon WR, Deng P, Budisavljevic MN, Syn WK, Wang C, Lipschutz JH, Kwon SH, Nihalnani D. The motor protein Myo1c regulates transforming growth factor-beta-signaling and fibrosis in podocytes. Kidney Int 96: 139–158, 2019. doi: 10.1016/j.kint.2019.02.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Fan Y, Eswarappa SM, Hitomi M, Fox PL. Myo1c facilitates G-actin transport to the leading edge of migrating endothelial cells. J Cell Biol 198: 47–55, 2012. doi: 10.1083/jcb.201111088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Venit T, Dzijak R, Kalendová A, Kahle M, Rohožková J, Schmidt V, Rulicke T, Rathkolb B, Hans W, Bohla A, Eickelberg O, Stoeger T, Wolf E, Yildirim AO, Gailus-Durner V, Fuchs H, de Angelis MH, Hozak P . Mouse nuclear myosin I knock-out shows interchangeability and redundancy of myosin isoforms in the cell nucleus. PLoS One 8: e61406, 2013. doi: 10.1371/journal.pone.0061406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Philimonenko VV, Zhao J, Iben S, Dingová H, Kyselá K, Kahle M, Zentgraf H, Hofmann WA, de Lanerolle P, Hozak P, Grummt I. Nuclear actin and myosin I are required for RNA polymerase I transcription. Nat Cell Biol 6: 1165–1172, 2004. doi: 10.1038/ncb1190. [DOI] [PubMed] [Google Scholar]
  • 14.Zattelman L, Regev R, Ušaj M, Reinke PYA, Giese S, Samson AO, Taft MH, Manstein DJ, Hein A. N-terminal splicing extensions of the human MYO1C gene fine-tune the kinetics of the three full-length myosin IC isoforms. J Biol Chem 292: 17804–17818, 2017. doi: 10.1074/jbc.M117.794008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Herrmann J, Gressner AM, Weiskirchen R. Immortal hepatic stellate cell lines: useful tools to study hepatic stellate cell biology and function? J Cellular Mol Med 11: 704–722, 2007. p doi: 10.1111/j.1582-4934.2007.00060.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Syn W–K, Jung Y, Omenetti A, Abdelmalek M, Guy CD, Yang L, Wang J, Witek RP, Fearing CM, Pereira TA, Teaberry V, Conde-Vancells J, Karaca GF, Diehl AM. Hedgehog-mediated epithelial-to-mesenchymal transition and fibrogenic repair in nonalcoholic fatty liver disease. Gastroenterology 137: 1478–1488, 2009. doi: 10.1053/j.gastro.2009.06.051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Lombardi AA, Gibb AA, Arif E, Kolmetzky DW, Tomar D, Luongo TS, Jadiya P, Murray EK, Lorkiewicz PK, Hajnoczky G, Murphy E, Arany ZP, Kelly DP, Margulies KB, Hill BG, Elrod JW. Mitochondrial calcium exchange links metabolism with the epigenome to control cellular differentiation. Nat Commun 10: 4509, 2019. doi: 10.1038/s41467-019-12103-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Chinthalapudi K, Taft MH, Martin R, Heissler SM, Preller M, Hartmann FK, Brandstaetter H, Kendrick-Jones J, Tsiavaliaris G, Gutzeit HO, Fedorov R, Buss F, Knolker H-J, Coluccio LM, Mastein D, Elrod JW. Mechanism and specificity of pentachloropseudilin-mediated inhibition of myosin motor activity. J Biol Chem 286: 29700–29708, 2011. doi: 10.1074/jbc.M111.239210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Arffa ML, Zapf MA, Kothari AN, Chang V, Gupta GN, Ding X, Al-Gayyar MM, Syn W, Elsherbiny NM, Kuo PC, Mi Z. Epigallocatechin-3-gallate upregulates miR-221 to inhibit osteopontin-dependent hepatic fibrosis. PLoS One 11: e0167435, 2016. doi: 10.1371/journal.pone.0167435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Choi SS, Syn W-K, Karaca GF, Omenetti A, Moylan CA, Witek RP, Agboola KM, Jung Y, Michelotti GA, Diehl AM, Murphy E. Leptin promotes the myofibroblastic phenotype in hepatic stellate cells by activating the hedgehog pathway. J Biol Chem 285: 36551–36560, 2010. doi: 10.1074/jbc.M110.168542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Omenetti A, Syn W-K, Jung Y, Francis H, Porrello A, Witek RP, Choi SS, Yang L, Mayo MJ, Gershwin ME, Alpini G, Diehl AM. Repair-related activation of hedgehog signaling promotes cholangiocyte chemokine production. Hepatology 50: 518–527, 2009. doi: 10.1002/hep.23019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Rockey DC, Housset CN, Friedman SL. Activation-dependent contractility of rat hepatic lipocytes in culture and in vivo. J Clin Invest 92: 1795–1804, 1993. doi: 10.1172/JCI116769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Dai C, Xiao X, Li D, Tun S, Wang Y, Velkov T, Teng S. Chloroquine ameliorates carbon tetrachloride-induced acute liver injury in mice via the concomitant inhibition of inflammation and induction of apoptosis. Cell Death Dis 9: 1164, 2018. doi: 10.1038/s41419-018-1136-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Ma X, Zhou Y, Qiao B, Jiang S, Shen Q, Han Y, Liu A, Chen X, Wei L, Zhou L, Zhao J. Androgen aggravates liver fibrosis by activation of NLRP3 inflammasome in CCl4-induced liver injury mouse model. Am J Physiol Endocrinol Metab 318: E817–E829, 2020. doi: 10.1152/ajpendo.00427.2019. [DOI] [PubMed] [Google Scholar]
  • 25.Khetchoumian K, Teletin M, Tisserand J, Mark M, Herquel B, Ignat M, Zucman-Rossi J, Cammas F, Lerouge T, Thibault C, Metzger, Chambon P, Losson R. Loss of Trim24 (Tif1alpha) gene function confers oncogenic activity to retinoic acid receptor alpha. Nat Genet 39: 1500–1506, 2007. doi: 10.1038/ng.2007.15. [DOI] [PubMed] [Google Scholar]
  • 26.Jiang S, Minter LC, Stratton SA, Yang P, Abbas HA, Akdemir ZC, Pant V, Post S, Gagea M, Lee RG, Lozano G, Barton MC. TRIM24 suppresses development of spontaneous hepatic lipid accumulation and hepatocellular carcinoma in mice. J Hepatol 62: 371–379, 2015. doi: 10.1016/j.jhep.2014.09.026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Moylan CA, Pang H, Dellinger A, Suzuki A, Garrett ME, Guy CD, Murphy SK, Ashley-Koch AE, Choi SS, Michaelotti GA, Hampton DD, Chen Y, Tillmann HL, Hauser MA, Abdelmalek MF, Diehl AM. Hepatic gene expression profiles differentiate presymptomatic patients with mild versus severe nonalcoholic fatty liver disease. Hepatology 59: 471–482, 2014. doi: 10.1002/hep.26661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Machado MV, Michelotti GA, Xie G, de Almeida TP, Boursier J, Bohnic B, Guy CD, Diehl AM. Mouse models of diet-induced nonalcoholic steatohepatitis reproduce the heterogeneity of the human disease. PLoS One 10: e0127991, 2015. doi: 10.1371/journal.pone.0127991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Chen HJ, Liu J. Actein ameliorates hepatic steatosis and fibrosis in high fat diet-induced NAFLD by regulation of insulin and leptin resistant. Biomed Pharmacother 97: 1386–1396, 2018. doi: 10.1016/j.biopha.2017.09.093. [DOI] [PubMed] [Google Scholar]
  • 30.Mori Y, Hinchcliff M, Wu M, Warner-Blankenship M, Lyons KM, Varga J. Connective tissue growth factor/CCN2-null mouse embryonic fibroblasts retain intact transforming growth factor-beta responsiveness. Exp Cell Res 314: 1094–1104, 2008. doi: 10.1016/j.yexcr.2007.12.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Tang L-Y, Heller M, Meng Z, Yu L-R, Tang Y, Zhou M, Zhang YE. Transforming growth factor-beta (TGF-beta) directly activates the JAK1-STAT3 axis to induce hepatic fibrosis in coordination with the SMAD pathway. J Biol Chem 292: 4302–4312, 2017. doi: 10.1074/jbc.M116.773085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Higashi T, Friedman SL, Hoshida Y. Hepatic stellate cells as key target in liver fibrosis. Adv Drug Deliv Rev 121: 27–42, 2017. doi: 10.1016/j.addr.2017.05.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Toyama T, Lee HC, Koga H, Wands JR, Kim M. Noncanonical Wnt11 inhibits hepatocellular carcinoma cell proliferation and migration. Mol Cancer Res 8: 254–265, 2010. p doi: 10.1158/1541-7786.MCR-09-0238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Brandstaetter H, Kishi-Itakura C, Tumbarello DA, Manstein DJ, Buss F. Loss of functional MYO1C/myosin 1c, a motor protein involved in lipid raft trafficking, disrupts autophagosome-lysosome fusion. Autophagy 10: 2310–2323, 2014. doi: 10.4161/15548627.2014.984272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Xue M, Jackson CJ. Extracellular matrix reorganization during wound healing and its impact on abnormal scarring. Adv Wound Care (New Rochelle) 4: 119–136, 2015. doi: 10.1089/wound.2013.0485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Rockey DC, Du Q, Shi Z. Smooth muscle alpha-actin deficiency leads to decreased liver fibrosis via impaired cytoskeletal signaling in hepatic stellate cells. Am J Pathol 189: 2209–2220, 2019. doi: 10.1016/j.ajpath.2019.07.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Gonzalez-Andrades M, Alonso-Pastor L, Mauris J, Cruzat A, Dohlman CH, Argüeso P. Establishment of a novel in vitro model of stratified epithelial wound healing with barrier function. Sci Rep 6: 19395, 2016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Vernon RB, Gooden MD. An improved method for the collagen gel contraction assay. In Vitro Cell Dev Biol Anim 38: 97–101, 2002. doi:. [DOI] [PubMed] [Google Scholar]
  • 39.Kobayashi T, Liu X, Kim HJ, Kohyama T, Wen F-Q, Abe S, Fang Q, Zhu YK, Spurzem JR, Bitterman P, Rennard SI. TGF-beta1 and serum both stimulate contraction but differentially affect apoptosis in 3D collagen gels. Respir Res 6: 141, 2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Greenwel P, Schwartz M, Rosas M, Peyrol S, Grimaud JA, Rojkind M. Characterization of fat-storing cell lines derived from normal and CCl4-cirrhotic livers. Differences in the production of interleukin-6. Lab Invest 65: 644–653, 1991. [PubMed] [Google Scholar]
  • 41.Carpino G, Morini S, Ginannicorradini S, Franchitto A, Merli M, Siciliano M, Gentilli F, Muda AO, Berloco P, Rossi M, Attili AF, Gaudio E. Alpha-SMA expression in hepatic stellate cells and quantitative analysis of hepatic fibrosis in cirrhosis and in recurrent chronic hepatitis after liver transplantation. Dig Liver Dis 37: 349–356, 2005. doi: 10.1016/j.dld.2004.11.009. [DOI] [PubMed] [Google Scholar]
  • 42.Bose A, Guilherme A, Robida SI, Nicoloro SMC, Zhou QL, Jiang ZY, Pomerleau DP, Czeck MP. Glucose transporter recycling in response to insulin is facilitated by myosin Myo1c. Nature 420: 821–824, 2002. doi: 10.1038/nature01246. [DOI] [PubMed] [Google Scholar]
  • 43.Hou W, Syn WK. Role of metabolism in hepatic stellate cell activation and fibrogenesis. Front Cell Dev Biol 6: 150, 2018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Tiwari A, Jung J-J, Inamdar SM, Nihalani D, Choudhury A. The myosin motor Myo1c is required for VEGFR2 delivery to the cell surface and for angiogenic signaling. Am J Physiol Heart Circ Physiol 304: H687–H696, 2013. doi: 10.1152/ajpheart.00744.2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Arif E, Sharma P, Solanki A, Mallik L, Rathore YS, Twal WO, Nath SK, Gandhi D, Holzman LB, Ostap EM, Nihalani D. Structural analysis of the Myo1c and Neph1 complex provides insight into the intracellular movement of Neph1. Mol Cell Biol 36: 1639–1654, 2016.doi: 10.1128/MCB.00020-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Takenouchi Y, Kitakaze K, Tsuboi K, Okamoto Y. Growth differentiation factor 15 facilitates lung fibrosis by activating macrophages and fibroblasts. Exp Cell Res 391: 112010, 2020.doi: 10.1016/j.yexcr.2020.112010. [DOI] [PubMed] [Google Scholar]

Articles from American Journal of Physiology - Gastrointestinal and Liver Physiology are provided here courtesy of American Physiological Society

RESOURCES