Publications by authors named "Ming-Jer Tang"

84 Publications

A de novo COL17A1 splice-site mutation causing a 7-bp deletion in a Taiwanese patient with junctional epidermolysis bullosa.

Eur J Dermatol 2021 Apr;31(2):267-269

Department of Dermatology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan, International Center for Wound Repair and Regeneration (iWRR), National Cheng Kung University, Tainan, Taiwan.

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http://dx.doi.org/10.1684/ejd.2021.4011DOI Listing
April 2021

Symmetry breaking of tissue mechanics in wound induced hair follicle regeneration of laboratory and spiny mice.

Nat Commun 2021 05 10;12(1):2595. Epub 2021 May 10.

Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA.

Tissue regeneration is a process that recapitulates and restores organ structure and function. Although previous studies have demonstrated wound-induced hair neogenesis (WIHN) in laboratory mice (Mus), the regeneration is limited to the center of the wound unlike those observed in African spiny (Acomys) mice. Tissue mechanics have been implicated as an integral part of tissue morphogenesis. Here, we use the WIHN model to investigate the mechanical and molecular responses of laboratory and African spiny mice, and report these models demonstrate opposing trends in spatiotemporal morphogenetic field formation with association to wound stiffness landscapes. Transcriptome analysis and K14-Cre-Twist1 transgenic mice show the Twist1 pathway acts as a mediator for both epidermal-dermal interactions and a competence factor for periodic patterning, differing from those used in development. We propose a Turing model based on tissue stiffness that supports a two-scale tissue mechanics process: (1) establishing a morphogenetic field within the wound bed (mm scale) and (2) symmetry breaking of the epidermis and forming periodically arranged hair primordia within the morphogenetic field (μm scale). Thus, we delineate distinct chemo-mechanical events in building a Turing morphogenesis-competent field during WIHN of laboratory and African spiny mice and identify its evo-devo advantages with perspectives for regenerative medicine.
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http://dx.doi.org/10.1038/s41467-021-22822-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8110808PMC
May 2021

PPARγ activation improves the microenvironment of perivascular adipose tissue and attenuates aortic stiffening in obesity.

J Biomed Sci 2021 Mar 29;28(1):22. Epub 2021 Mar 29.

Institute of Clinical Medicine, National Cheng Kung University, Tainan, Taiwan, ROC.

Background: Obesity-related cardiovascular risk, end points, and mortality are strongly related to arterial stiffening. Current therapeutic approaches for arterial stiffening are not focused on direct targeting within the vessel. Perivascular adipose tissue (PVAT) surrounding the artery has been shown to modulate vascular function and inflammation. Peroxisome proliferator-activated receptor γ (PPARγ) activation significantly decreases arterial stiffness and inflammation in diabetic patients with coronary artery disease. Thus, we hypothesized that PPARγ activation alters the PVAT microenvironment, thereby creating a favorable environment for the attenuation of arterial stiffening in obesity.

Methods: Obese ob/ob mice were used to investigate the effect of PPARγ activation on the attenuation of arterial stiffening. Various cell types, including macrophages, fibroblasts, adipocytes, and vascular smooth muscle cells, were used to test the inhibitory effect of pioglitazone, a PPARγ agonist, on the expression of elastolytic enzymes.

Results: PPARγ activation by pioglitazone effectively attenuated arterial stiffening in ob/ob mice. This beneficial effect was not associated with the repartitioning of fat from or changes in the browning of the PVAT depot but was strongly related to improvement of the PVAT microenvironment, as evidenced by reduction in the expression of pro-inflammatory and pro-oxidative factors. Pioglitazone treatment attenuated obesity-induced elastin fiber fragmentation and elastolytic activity and ameliorated the obesity-induced upregulation of cathepsin S and metalloproteinase 12, predominantly in the PVAT. In vitro, pioglitazone downregulated Ctss and Mmp12 in macrophages, fibroblasts, and adipocytes-cell types residing within the adventitia and PVAT. Ultimately, several PPARγ binding sites were found in Ctss and Mmp12 in Raw 264.7 and 3T3-L1 cells, suggesting a direct regulatory mechanism by which PPARγ activation repressed the expression of Ctss and Mmp-12 in macrophages and fibroblasts.

Conclusions: PPARγ activation attenuated obesity-induced arterial stiffening and reduced the inflammatory and oxidative status of PVAT. The improvement of the PVAT microenvironment further contributed to the amelioration of elastin fiber fragmentation, elastolytic activity, and upregulated expression of Ctss and Mmp12. Our data highlight the PVAT microenvironment as an important target against arterial stiffening in obesity and provide a novel strategy for the potential clinical use of PPARγ agonists as a therapeutic against arterial stiffness through modulation of PVAT function.
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http://dx.doi.org/10.1186/s12929-021-00720-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8008548PMC
March 2021

VEGF-Induced Endothelial Podosomes via ROCK2-Dependent Thrombomodulin Expression Initiate Sprouting Angiogenesis.

Arterioscler Thromb Vasc Biol 2021 05 18;41(5):1657-1671. Epub 2021 Mar 18.

Department of Biochemistry and Molecular Biology, National Cheng Kung University, Tainan, Taiwan (C.-H.K., P.-K.C., G.-Y.S., H.-L.W.)

[Figure: see text].
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http://dx.doi.org/10.1161/ATVBAHA.121.315931DOI Listing
May 2021

Pancreatic stellate cells activated by mutant KRAS-mediated PAI-1 upregulation foster pancreatic cancer progression via IL-8.

Theranostics 2019 23;9(24):7168-7183. Epub 2019 Sep 23.

Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan.

The dense fibrotic stroma enveloping pancreatic tumors is a major cause of drug resistance. Pancreatic stellate cells (PSCs) in the stroma can be activated to induce intra-tumor fibrosis and worsen patient survival; however, the molecular basics for the regulation of PSC activation remains unclear. The coculture system was used to study cancer cell-PSC interactions. Atomic force microscopy was used to measure the stiffness of tumor tissues and coculture gels. Cytokine arrays, qPCR, and Western blotting were performed to identify the potential factors involved in PSC activation and to elucidate underlying pathways. PSC activation characterized by α-SMA expression was associated with increased pancreatic tumor stiffness and poor prognosis. Coculture with cancer cells induced PSC activation, which increased organotypic coculture gel stiffness and cancer cell invasion. Cancer cells-derived PAI-1 identified from coculture medium could activate PSCs, consistent with pancreatic cancer tissue microarray analysis showing a strong positive correlation between PAI-1 and α-SMA expression. Suppression by knocking down PAI-1 in cancer cells demonstrated the requirement of PAI-1 for coculture-induced PSC activation and gel stiffness. PAI-1 could be upregulated by KRAS in pancreatic cancer cells through ERK. In PSCs, inhibition of LRP-1, ERK, and c-JUN neutralized the effect of PAI-1, suggesting the contribution of LRP-1/ERK/c-JUN signaling. Furthermore, activated PSCs might exacerbate malignant behavior of cancer cells via IL-8 because suppression of IL-8 signaling reduced pancreatic tumor growth and fibrosis . KRAS-mutant pancreatic cancer cells can activate PSCs through PAI-1/LRP-1 signaling to promote fibrosis and cancer progression.
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http://dx.doi.org/10.7150/thno.36830DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6831292PMC
September 2020

Dichotomy of the function of DDR1 in cells and disease progression.

Biochim Biophys Acta Mol Cell Res 2019 11 5;1866(11):118473. Epub 2019 Apr 5.

International Center for Wound Repair and Regeneration, Tainan, Taiwan; Department of Physiology, National Cheng Kung University, Tainan, Taiwan. Electronic address:

Discoidin domain receptors DDR1 and DDR2 are collagen receptor tyrosine kinases that have many roles in tissue development and disease progression. Under physiological conditions, DDR1 is predominantly expressed in epithelial cells and functions to maintain cell differentiation and tissue homeostasis. A switch in expression from DDR1 to DDR2 occurs during epithelial-to-mesenchymal transition. However, opposite effects of DDR1 are reported to be involved in the progression of cancer and fibrotic diseases. Accumulating evidence suggests that DDR1 is involved in pro-metastasis and pro-survival signals. This review summarizes the roles of DDR1 in epithelial cell differentiation, cell migration, cancer progression and tissues fibrosis and highlights how the dichotomous functions of DDR1 may relevant to different cell types and statues. Elucidation of the underlying mechanism of the dichotomous functions of DDR1 will help to develop DDR1 as a therapeutic target.
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http://dx.doi.org/10.1016/j.bbamcr.2019.04.003DOI Listing
November 2019

Tks5 and Dynamin-2 enhance actin bundle rigidity in invadosomes to promote myoblast fusion.

J Cell Biol 2019 05 20;218(5):1670-1685. Epub 2019 Mar 20.

Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan

Skeletal muscle development requires the cell-cell fusion of differentiated myoblasts to form muscle fibers. The actin cytoskeleton is known to be the main driving force for myoblast fusion; however, how actin is organized to direct intercellular fusion remains unclear. Here we show that an actin- and dynamin-2-enriched protrusive structure, the invadosome, is required for the fusion process of myogenesis. Upon differentiation, myoblasts acquire the ability to form invadosomes through isoform switching of a critical invadosome scaffold protein, Tks5. Tks5 directly interacts with and recruits dynamin-2 to the invadosome and regulates its assembly around actin filaments to strengthen the stiffness of dynamin-actin bundles and invadosomes. These findings provide a mechanistic framework for the acquisition of myogenic fusion machinery during myogenesis and reveal a novel structural function for Tks5 and dynamin-2 in organizing actin filaments in the invadosome to drive membrane fusion.
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http://dx.doi.org/10.1083/jcb.201809161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6504888PMC
May 2019

Coincidence Detection of Membrane Stretch and Extracellular pH by the Proton-Sensing Receptor OGR1 (GPR68).

Curr Biol 2018 12 21;28(23):3815-3823.e4. Epub 2018 Nov 21.

Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, UK. Electronic address:

The physical environment critically affects cell shape, proliferation, differentiation, and survival by exerting mechanical forces on cells. These forces are sensed and transduced into intracellular signals and responses by cells. A number of different membrane and cytoplasmic proteins have been implicated in sensing mechanical forces, but the picture is far from complete, and the exact transduction pathways remain largely elusive. Furthermore, mechanosensation takes place alongside chemosensation, and cells need to integrate physical and chemical signals to respond appropriately and ensure normal tissue and organ development and function. Here, we report that ovarian cancer G protein coupled receptor 1 (OGR1) (aka GPR68) acts as coincidence detector of membrane stretch and its physiological ligand, extracellular H. Using fluorescence imaging, substrates of different stiffness, microcontact printing methods, and cell-stretching techniques, we show that OGR1 only responds to extracellular acidification under conditions of membrane stretch and vice versa. The level of OGR1 activity mirrors the extent of membrane stretch and degree of extracellular acidification. Furthermore, actin polymerization in response to membrane stretch is critical for OGR1 activity, and its depolymerization limits how long OGR1 remains responsive following a stretch event, thus providing a "memory" for past stretch. Cells experience changes in membrane stretch and extracellular pH throughout their lifetime. Because OGR1 is a widely expressed receptor, it represents a unique yet widespread mechanism that enables cells to respond dynamically to mechanical and pH changes in their microenvironment by integrating these chemical and physical stimuli at the receptor level.
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http://dx.doi.org/10.1016/j.cub.2018.10.046DOI Listing
December 2018

Mechanical forces in skin disorders.

J Dermatol Sci 2018 Jun 8;90(3):232-240. Epub 2018 Mar 8.

Department of Dermatology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan; International Research Center for Wound Repair and Regeneration, National Cheng Kung University, Tainan, Taiwan. Electronic address:

Mechanical forces are known to regulate homeostasis of the skin and play a role in the pathogenesis of skin diseases. The epidermis consists of keratinocytes that are tightly adhered to each other by cell junctions. Defects in keratins or desmosomal/hemidesmosomal proteins lead to the attenuation of mechanical strength and formation of intraepidermal blisters in the case of epidermolysis bullosa simplex. The dermis is rich in extracellular matrix, especially collagen, and provides the majority of tensile force in the skin. Keloid and hypertrophic scar, which is the result of over-production of collagen by fibroblasts during the wound healing, are associated with extrinsic tensile forces and changes of intrinsic mechanical properties of the cell. Increasing evidences shows that stiffness of the skin environment determines the regenerative ability during wound healing process. Mechanotransduction pathways are also involved in the morphogenesis and cyclic growth of hair follicles. The development of androgenetic alopecia is correlated to tensile forces generated by the fibrous tissue underlying the scalp. Acral melanoma predominantly occurs in the weight-bearing area of the foot suggesting the role of mechanical stress. Increased dermal stiffness from fibrosis might be the cause of recessive dystrophic epidermolysis bullosa associated squamous cell carcinoma. Strategies to change the mechanical forces or modify the mechanotransduction signals may lead to a new way to treat skin diseases and promote skin regeneration.
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http://dx.doi.org/10.1016/j.jdermsci.2018.03.004DOI Listing
June 2018

Bcl2 regulates store-operated Ca entry to modulate ER stress-induced apoptosis.

Cell Death Discov 2018 Dec 26;4:37. Epub 2018 Feb 26.

2Institute of Basic Medical Sciences, National Cheng Kung University, Tainan, 701 Taiwan.

Ca plays a significant role in linking the induction of apoptosis. The key anti-apoptotic protein, Bcl-2, has been reported to regulate the movement of Ca across the ER membrane, but the exact effect of Bcl-2 on Ca levels remains controversial. Store-operated Ca entry (SOCE), a major mode of Ca uptake in non-excitable cells, is activated by depletion of Ca in the ER. Depletion of Ca in the ER causes translocation of the SOC channel activator, STIM1, to the plasma membrane. Thereafter, STIM1 binds to Orai1 or/and TRPC1 channels, forcing them to open and thereby allow Ca entry. In addition, several anti-cancer drugs have been reported to induce apoptosis of cancer cells via the SOCE pathway. However, the detailed mechanism underlying the regulation of SOCE by Bcl-2 is not well understood. In this study, a three-amino acid mutation within the Bcl-2 BH1 domain was generated to verify the role of Bcl-2 in Ca handling during ER stress. The subcellular localization of the Bcl-2 mutant (mt) is similar to that in the wild-type Bcl-2 (WT) in the ER and mitochondria. We found that mt enhanced thapsigargin and tunicamycin-induced apoptosis through ER stress-mediated apoptosis but not through the death receptor- and mitochondria-dependent apoptosis, while WT prevented thapsigargin- and tunicamycin-induced apoptosis. In addition, mt depleted Ca in the ER lumen and also increased the expression of SOCE-related molecules. Therefore, a massive Ca influx via SOCE contributed to caspase activation and apoptosis. Furthermore, inhibiting SOCE or chelating either extracellular or intracellular Ca inhibited mt-mediated apoptosis. In brief, our results explored the critical role of Bcl-2 in Ca homeostasis and the modulation of ER stress.
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http://dx.doi.org/10.1038/s41420-018-0039-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5841437PMC
December 2018

Caveolin-1 down-regulation is required for Wnt5a-Frizzled 2 signalling in Ha-Ras -induced cell transformation.

J Cell Mol Med 2018 05 4;22(5):2631-2643. Epub 2018 Mar 4.

Department of Physiology, National Cheng Kung University, Tainan, Taiwan.

Caveolin-1 (Cav1) is down-regulated during MK4 (MDCK cells harbouring inducible Ha-Ras gene) transformation by Ha-Ras . Cav1 overexpression abrogates the Ha-Ras -driven transformation of MK4 cells; however, the targeted down-regulation of Cav1 is not sufficient to mimic this transformation. Cav1-silenced cells, including MK4/shCav1 cells and MDCK/shCav1 cells, showed an increased cell area and discontinuous junction-related proteins staining. Cellular and mechanical transformations were completed when MDCK/shCav1 cells were treated with medium conditioned by MK4 cells treated with IPTG (MK4+I-CM) but not with medium conditioned by MK4 cells. Nanoparticle tracking analysis showed that Ha-Ras -inducing MK4 cells increased exosome-like microvesicles release compared with their normal counterparts. The cellular and mechanical transformation activities of MK4+I-CM were abolished after heat treatment and exosome depletion and were copied by exosomes derived from MK4+I-CM (MK4+I-EXs). Wnt5a, a downstream product of Ha-Ras , was markedly secreted by MK4+I-CM and MK4+I-EXs. Suppression of Wnt5a expression and secretion using the porcupine inhibitor C59 or Wnt5a siRNA inhibited the Ha-Ras - and MK4+I-CM-induced transformation of MK4 cells and MDCK/shCav1 cells, respectively. Cav1 down-regulation, either by Ha-Ras or targeted shRNA, increased frizzled-2 (Fzd2) protein levels without affecting its mRNA levels, suggesting a novel role of Cav1 in negatively regulating Fzd2 expression. Additionally, silencing Cav1 facilitated the internalization of MK4+I-EXs in MDCK cells. These data suggest that Cav1-dependent repression of Fzd2 and exosome uptake is potentially relevant to its antitransformation activity, which hinders the activation of Ha-Ras -Wnt5a-Stat3 pathway. Altogether, these results suggest that both decreasing Cav1 and increasing exosomal Wnt5a must be implemented during Ha-Ras -driven cell transformation.
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http://dx.doi.org/10.1111/jcmm.13531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5908114PMC
May 2018

Mechanotransduction of matrix stiffness in regulation of focal adhesion size and number: reciprocal regulation of caveolin-1 and β1 integrin.

Sci Rep 2017 11 8;7(1):15008. Epub 2017 Nov 8.

Department of Physiology, National Cheng Kung University, Tainan, Taiwan.

Focal adhesion (FA) assembly, mediated by integrin activation, responds to matrix stiffness; however, the underlying mechanisms are unclear. Here, we showed that β1 integrin and caveolin-1 (Cav1) levels were decreased with declining matrix stiffness. Soft matrix selectively downregulated β1 integrin by endocytosis and subsequent lysosomal degradation. Disruption of lipid rafts with methyl-β-cyclodextrin or nystatin, or knockdown of Cav1 by siRNA decreased cell spreading, FA assembly, and β1 integrin protein levels in cells cultured on stiff matrix. Overexpression of Cav1, particularly the phospho-mimetic mutant Cav1-Y14D, averted soft matrix-induced decreases in β1 integrin protein levels, cell spreading, and FA assembly in NMuMG cells. Interestingly, overexpression of an auto-clustering β1 integrin hindered soft matrix-induced reduction of Cav1 and cell spreading, which suggests a reciprocal regulation between β1 integrin and Cav1. Finally, co-expression of this auto-clustering β1 integrin and Cav1-Y14D synergistically enhanced cell spreading, and FA assembly in HEK293T cells cultured on either stiff ( > G Pa) or soft (0.2 kPa) matrices. Collectively, these results suggest that matrix stiffness governs the expression of β1 integrin and Cav1, which reciprocally control each other, and subsequently determine FA assembly and turnover.
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http://dx.doi.org/10.1038/s41598-017-14932-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5678369PMC
November 2017

The tension biology of wound healing.

Exp Dermatol 2019 04 20;28(4):464-471. Epub 2017 Dec 20.

International Research Center of Wound Repair and Regeneration (iWRR), National Cheng Kung University, Tainan, Taiwan.

Following skin wounding, the healing outcome can be: regeneration, repair with normal scar tissue, repair with hypertrophic scar tissue or the formation of keloids. The role of chemical factors in wound healing has been extensively explored, and while there is evidence suggesting the role of mechanical forces, its influence is much less well defined. Here, we provide a brief review on the recent progress of the role of mechanical force in skin wound healing by comparing laboratory mice, African spiny mice, fetal wound healing and adult scar keloid formation. A comparison across different species may provide insight into key regulators. Interestingly, some findings suggest tension can induce an immune response, and this provides a new link between mechanical and chemical forces. Clinically, manipulating skin tension has been demonstrated to be effective for scar prevention and treatment, but not for tissue regeneration. Utilising this knowledge, specialists may modulate regulatory factors and develop therapeutic strategies to reduce scar formation and promote regeneration.
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http://dx.doi.org/10.1111/exd.13460DOI Listing
April 2019

Caveolin-1 Controls Hyperresponsiveness to Mechanical Stimuli and Fibrogenesis-Associated RUNX2 Activation in Keloid Fibroblasts.

J Invest Dermatol 2018 01 9;138(1):208-218. Epub 2017 Sep 9.

International Research Center of Wound Repair and Regeneration, National Cheng Kung University, Tainan, Taiwan; Department of Physiology, College of Medicine, National Cheng Kung University, Tainan, Taiwan. Electronic address:

Keloids are pathological scars characterized by excessive extracellular matrix production that are prone to form in body sites with increased skin tension. CAV1, the principal coat protein of caveolae, has been associated with the regulation of cell mechanics, including cell softening and loss of stiffness sensing ability in NIH3T3 fibroblasts. Although CAV1 is present in low amounts in keloid fibroblasts (KFs), the causal association between CAV1 down-regulation and its aberrant responses to mechanical stimuli remain unclear. In this study, atomic force microscopy showed that KFs were softer than normal fibroblasts with a loss of stiffness sensing. The decrease of CAV1 contributed to the hyperactivation of fibrogenesis-associated RUNX2, a transcription factor germane to osteogenesis/chondrogenesis, and increased migratory ability in KFs. Treatment of KFs with trichostatin A, which increased the acetylation level of histone H3, increased CAV1 and decreased RUNX2 and fibronectin. Trichostatin A treatment also resulted in cell stiffening and decreased migratory ability in KFs. Collectively, these results suggest a role for CAV1 down-regulation in linking the aberrant responsiveness to mechanical stimulation and extracellular matrix accumulation with the progression of keloids, findings that may lead to new developments in the prevention and treatment of keloid scarring.
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http://dx.doi.org/10.1016/j.jid.2017.05.041DOI Listing
January 2018

Vibrio vulnificus MARTX cytotoxin causes inactivation of phagocytosis-related signaling molecules in macrophages.

J Biomed Sci 2017 Aug 19;24(1):58. Epub 2017 Aug 19.

Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, 70101, Taiwan.

Background: Vibrio vulnificus is a marine bacterial species that causes opportunistic infections manifested by serious skin lesions and fulminant septicemia in humans. We have previously shown that the multifunctional autoprocessing repeats in toxin (MARTX) of a biotype 1 V. vulnificus strain promotes survival of this organism in the host by preventing it from engulfment by the phagocytes. The purpose of this study was to further explore how MARTX inhibits phagocytosis of this microorganism by the macrophage.

Methods: We compared between a wild-type V. vulnificus strain and its MARTX-deficient mutant for a variety of phagocytosis-related responses, including morphological change and activation of signaling molecules, they induced in the macrophage. We also characterized a set of MARTX domain-deletion mutants to define the regions associated with antiphagocytosis activity.

Results: The RAW 264.7 cells and mouse peritoneal exudate macrophages underwent cell rounding accompanied by F-actin disorganization in the presence of MARTX. In addition, phosphorylation of some F-actin rearrangement-associated signaling molecules, including Lyn, Fgr and Hck of the Src family kinases (SFKs), focal adhesion kinase (FAK), proline-rich tyrosine kinase 2 (Pyk2), phosphoinositide 3-kinase (PI3K) and Akt, but not p38, was decreased. By using specific inhibitors, we found that these kinases were all involved in the phagocytosis of MARTX-deficient mutant in an order of SFKs-FAK/Pyk2-PI3K-Akt. Deletion of the effector domains in the central region of MARTX could lead to reduced cytotoxicity, depending on the region and size of deletion, but did not affect the antiphagocytosis activity and ability to cause rounding of macrophage. Reduced phosphorylation of Akt was closely associated with inhibition of phagocytosis by the wild-type strain and MARTX domain-deletion mutants, and expression of the constitutively active Akt, myr-Akt, enhanced the engulfment of these strains by macrophage.

Conclusions: MARTX could inactivate the SFKs-FAK/Pyk2-PI3K-Akt signaling pathway in the macrophages. This might lead to impaired phagocytosis of the V. vulnificus-infected macrophage. The majority of the central region of MARTX is not associated with the antiphagocytosis activity.
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http://dx.doi.org/10.1186/s12929-017-0368-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5563386PMC
August 2017

DDR1 promotes E-cadherin stability via inhibition of integrin-β1-Src activation-mediated E-cadherin endocytosis.

Sci Rep 2016 11 8;6:36336. Epub 2016 Nov 8.

Institute of Basic Medical Sciences, National Cheng Kung University, Tainan, Taiwan.

Discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase of collagen, is primarily expressed in epithelial cells. Activation of DDR1 stabilises E-cadherin located on the cell membrane; however, the detailed mechanism of DDR1-stabilised E-cadherin remains unclear. We performed DDR1 knockdown (Sh-DDR1) on Mardin-Darby canine kidney cells to investigate the mechanism of DDR1-stabilised E-cadherin. Sh-DDR1 decreased junctional localisation, increased endocytosis of E-cadherin, and increased physical interactions between E-cadherin and clathrin. Treatment of the dynamin inhibitor Dyngo 4a suppressed Sh-DDR1-induced E-cadherin endocytosis. In addition, the phosphorylation level of Src tyrosine 418 was increased in Sh-DDR1 cell junctions, and inhibition of Src activity decreased Sh-DDR1-induced E-cadherin endocytosis. To characterise the molecular mechanisms, blocking integrin β1 decreased Src activity and E-cadherin junctional localisation in Sh-DDR1 cells. Photoconversion results showed that inhibition of Src activity rescued E-cadherin membrane stability and that inhibition of integrin β1-Src signalling decreased stress fibres and rescued E-cadherin membrane stability in Sh-DDR1 cells. Taken together, DDR1 stabilised membrane localisation of E-cadherin by inhibiting the integrin β1-Src-mediated clathrin-dependent endocytosis pathway.
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http://dx.doi.org/10.1038/srep36336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5099905PMC
November 2016

Spatial distribution of filament elasticity determines the migratory behaviors of a cell.

Cell Adh Migr 2016 07 26;10(4):368-77. Epub 2016 Feb 26.

a Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University , Tainan , Taiwan.

Any cellular response leading to morphological changes is highly tuned to balance the force generated from structural reorganization, provided by actin cytoskeleton. Actin filaments serve as the backbone of intracellular force, and transduce external mechanical signal via focal adhesion complex into the cell. During migration, cells not only undergo molecular changes but also rapid mechanical modulation. Here we focus on determining, the role of spatial distribution of mechanical changes of actin filaments in epithelial, mesenchymal, fibrotic and cancer cells with non-migration, directional migration, and non-directional migration behaviors using the atomic force microscopy. We found 1) non-migratory cells only generated one type of filament elasticity, 2) cells generating spatially distributed two types of filament elasticity showed directional migration, and 3) pathologic cells that autonomously generated two types of filament elasticity without spatial distribution were actively migrating non-directionally. The demonstration of spatial regulation of filament elasticity of different cell types at the nano-scale highlights the coupling of cytoskeletal function with physical characters at the sub-cellular level, and provides new research directions for migration related disease.
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http://dx.doi.org/10.1080/19336918.2016.1156825DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986705PMC
July 2016

Role of Excessive Autophagy Induced by Mechanical Overload in Vein Graft Neointima Formation: Prediction and Prevention.

Sci Rep 2016 Feb 26;6:22147. Epub 2016 Feb 26.

Institute of Basic Medical Sciences, National Cheng Kung University, Tainan, Taiwan.

Little is known regarding the interplays between the mechanical and molecular bases for vein graft restenosis. We elucidated the stenosis initiation using a high-frequency ultrasonic (HFU) echogenicity platform and estimated the endothelium yield stress from von-Mises stress computation to predict the damage locations in living rats over time. The venous-arterial transition induced the molecular cascades for autophagy and apoptosis in venous endothelial cells (ECs) to cause neointimal hyperplasia, which correlated with the high echogenicity in HFU images and the large mechanical stress that exceeded the yield strength. The ex vivo perfusion of arterial laminar shear stress to isolated veins further confirmed the correlation. EC damage can be rescued by inhibiting autophagy formation using 3-methyladenine (3-MA). Pretreatment of veins with 3-MA prior to grafting reduced the pathological increases of echogenicity and neointima formation in rats. Therefore, this platform provides non-invasive temporal spatial measurement and prediction of restenosis after venous-arterial transition as well as monitoring the progression of the treatments.
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http://dx.doi.org/10.1038/srep22147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4768319PMC
February 2016

Albumin stimulates renal tubular inflammation through an HSP70-TLR4 axis in mice with early diabetic nephropathy.

Dis Model Mech 2015 Oct 6;8(10):1311-21. Epub 2015 Aug 6.

Institute of Basic Medical Sciences, National Cheng Kung University, Tainan 701, Taiwan Institute of Clinical Medicine, National Cheng Kung University, Tainan 701, Taiwan Research Center of Clinical Medicine, National Cheng Kung University Hospital, Tainan 704, Taiwan, Republic of China

Increased urinary albumin excretion is not simply an aftermath of glomerular injury, but is also involved in the progression of diabetic nephropathy (DN). Whereas Toll-like receptors (TLRs) are incriminated in the renal inflammation of DN, whether and how albumin is involved in the TLR-related renal inflammatory response remains to be clarified. Here, we showed that both TLR2 and TLR4, one of their putative endogenous ligands [heat shock protein 70 (HSP70)] and nuclear factor-κB promoter activity were markedly elevated in the kidneys of diabetic mice. A deficiency of TLR4 but not of TLR2 alleviated albuminuria, tubulointerstitial fibrosis and inflammation induced by diabetes. The protection against renal injury in diabetic Tlr4(-/-) mice was associated with reduced tubular injuries and preserved cubilin levels, rather than amelioration of glomerular lesions. In vitro studies revealed that albumin, a stronger inducer than high glucose (HG), induced the release of HSP70 from proximal tubular cells. HSP70 blockade ameliorated albumin-induced inflammatory mediators. HSP70 triggered the production of inflammatory mediators in a TLR4-dependent manner. Moreover, HSP70 inhibition in vivo ameliorated diabetes-induced albuminuria, inflammatory response and tubular injury. Finally, we found that individuals with DN had higher levels of TLR4 and HSP70 in the dilated tubules than non-diabetic controls. Thus, activation of the HSP70-TLR4 axis, stimulated at least in part by albumin, in the tubular cell is a newly identified mechanism associated with induction of tubulointerstitial inflammation and aggravation of pre-existing microalbuminuria in the progression of DN.
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http://dx.doi.org/10.1242/dmm.019398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4610229PMC
October 2015

Matrix-Stiffness-Regulated Inverse Expression of Krüppel-Like Factor 5 and Krüppel-Like Factor 4 in the Pathogenesis of Renal Fibrosis.

Am J Pathol 2015 Sep 26;185(9):2468-81. Epub 2015 Jul 26.

Institute of Basic Medical Sciences, National Cheng-Kung University Medical College, Tainan, Taiwan; Department of Physiology, National Cheng-Kung University Medical College, Tainan, Taiwan. Electronic address:

The proliferation of mouse proximal tubular epithelial cells in ex vivo culture depends on matrix stiffness. Combined analysis of the microarray and experimental data revealed that Krüppel-like factor (Klf)5 was the most up-regulated transcription factor accompanied by the down-regulation of Klf4 when cells were on stiff matrix. These changes were reversed by soft matrix via extracellular signal-regulated kinase (ERK) inactivation. Knockdown of Klf5 or forced expression of Klf4 inhibited stiff matrix-induced cell spreading and proliferation, suggesting that Klf5/Klf4 act as positive and negative regulators, respectively. Moreover, stiff matrix-activated ERK increased the protein level and nuclear translocation of mechanosensitive Yes-associated protein 1 (YAP1), which is reported to prevent Klf5 degradation. Finally, in vivo model of unilateral ureteral obstruction revealed that matrix stiffness-regulated Klf5/Klf4 is related to the pathogenesis of renal fibrosis. In the dilated tubules of obstructed kidney, ERK/YAP1/Klf5/cyclin D1 axis was up-regulated and Klf4 was down-regulated. Inhibition of collagen crosslinking by lysyl oxidase inhibitor alleviated unilateral ureteral obstruction-induced tubular dilatation and proliferation, preserved Klf4, and suppressed the ERK/YAP1/Klf5/cyclin D1 axis. This study unravels a novel mechanism how matrix stiffness regulates cellular proliferation and highlights the importance of matrix stiffness-modulated Klf5/Klf4 in the regulation of renal physiologic functions and fibrosis progression.
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http://dx.doi.org/10.1016/j.ajpath.2015.05.019DOI Listing
September 2015

Mechanical phenotype of cancer cells: cell softening and loss of stiffness sensing.

Oncotarget 2015 Aug;6(25):20946-58

Department of Physiology, National Cheng Kung University, Tainan, Taiwan.

The stiffness sensing ability is required to respond to the stiffness of the matrix. Here we determined whether normal cells and cancer cells display distinct mechanical phenotypes. Cancer cells were softer than their normal counterparts, regardless of the type of cancer (breast, bladder, cervix, pancreas, or Ha-RasV12-transformed cells). When cultured on matrices of varying stiffness, low stiffness decreased proliferation in normal cells, while cancer cells and transformed cells lost this response. Thus, cancer cells undergo a change in their mechanical phenotype that includes cell softening and loss of stiffness sensing. Caveolin-1, which is suppressed in many tumor cells and in oncogene-transformed cells, regulates the mechanical phenotype. Caveolin-1-upregulated RhoA activity and Y397FAK phosphorylation directed actin cap formation, which was positively correlated with cell elasticity and stiffness sensing in fibroblasts. Ha-RasV12-induced transformation and changes in the mechanical phenotypes were reversed by re-expression of caveolin-1 and mimicked by the suppression of caveolin-1 in normal fibroblasts. This is the first study to describe this novel role for caveolin-1, linking mechanical phenotype to cell transformation. Furthermore, mechanical characteristics may serve as biomarkers for cell transformation.
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http://dx.doi.org/10.18632/oncotarget.4173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4673241PMC
August 2015

Vimentin contributes to epithelial-mesenchymal transition cancer cell mechanics by mediating cytoskeletal organization and focal adhesion maturation.

Oncotarget 2015 Jun;6(18):15966-83

Institute of Basic Medical Sciences, National Cheng Kung University, Tainan, Taiwan.

Modulations of cytoskeletal organization and focal adhesion turnover correlate to tumorigenesis and epithelial-mesenchymal transition (EMT), the latter process accompanied by the loss of epithelial markers and the gain of mesenchymal markers (e.g., vimentin). Clinical microarray results demonstrated that increased levels of vimentin mRNA after chemotherapy correlated to a poor prognosis of breast cancer patients. We hypothesized that vimentin mediated the reorganization of cytoskeletons to maintain the mechanical integrity in EMT cancer cells. By using knockdown strategy, the results showed reduced cell proliferation, impaired wound healing, loss of directional migration, and increased large membrane extension in MDA-MB 231 cells. Vimentin depletion also induced reorganization of cytoskeletons and reduced focal adhesions, which resulted in impaired mechanical strength because of reduced cell stiffness and contractile force. In addition, overexpressing vimentin in MCF7 cells increased cell stiffness, elevated cell motility and directional migration, reoriented microtubule polarity, and increased EMT phenotypes due to the increased β1-integrin and the loss of junction protein E-cadherin. The EMT-related transcription factor slug was also mediated by vimentin. The current study demonstrated that vimentin serves as a regulator to maintain intracellular mechanical homeostasis by mediating cytoskeleton architecture and the balance of cell force generation in EMT cancer cells.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4599250PMC
http://dx.doi.org/10.18632/oncotarget.3862DOI Listing
June 2015

Mechanical coupling of cytoskeletal elasticity and force generation is crucial for understanding the migrating nature of keloid fibroblasts.

Exp Dermatol 2015 Aug 8;24(8):579-84. Epub 2015 May 8.

Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan.

One of the key features of keloid is its fibroblasts migrating beyond the original wound border. During migration, cells not only undergo molecular changes but also mechanical modulation. This process is led by actin filaments serving as the backbone of intra-cellular force and transduces external mechanical signal via focal adhesion complex into the cell. Here, we focus on determining the mechanical changes of actin filaments and the spatial distribution of forces in response to changing chemical stimulations and during cell migration. Atomic force microscopy and micropost array detector are used to determine and compare the magnitude and distribution of filament elasticity and force generation in fibroblasts and keloid fibroblasts. We found both filament elasticity and force generation show spatial distribution in a polarized and migrating cell. Such spatial distribution is disrupted when mechano-signalling is perturbed by focal adhesion kinase inhibitor and in keloid fibroblasts. The demonstration of keloid pathology at the nanoscale highlights the coupling of cytoskeletal function with physical characters at the subcellular level and provides new research directions for migration-related disease such as keloid.
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http://dx.doi.org/10.1111/exd.12731DOI Listing
August 2015

β-PIX controls intracellular viscoelasticity to regulate lung cancer cell migration.

J Cell Mol Med 2015 May 16;19(5):934-47. Epub 2015 Feb 16.

Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan.

Cancer metastasis occurs via a progress involving abnormal cell migration. Cell migration, a dynamic physical process, is controlled by the cytoskeletal system, which includes the dynamics of actin organization and cellular adhesive organelles, focal adhesions (FAs). However, it is not known whether the organization of actin cytoskeletal system has a regulatory role in the physiologically relevant aspects of cancer metastasis. In the present studies, it was found that lung adenocarcinoma cells isolated from the secondary lung cancer of the lymph nodes, H1299 cells, show specific dynamics in terms of the actin cytoskeleton and FAs. This results in a higher level of mobility and this is regulated by an immature FA component, β-PIX (PAK-interacting exchange factor-β). In H1299 cells, β-PIX's activity was found not to be down-regulated by sequestration onto stress fibres, as the cells did not bundle actin filaments into stress fibres. Thus, β-PIX mainly remained localized at FAs, which allowed maturation of nascent adhesions into focal complexes; this resulted in actin polymerization, increased actin network integrity, changes in the intracellular microrheology at the peripheral of the cell, and cell polarity, which in turn regulated cell migration. Perturbation of β-PIX caused an inhibition of cell migration, including migration velocity, accumulated distance and directional persistence. Our results demonstrate the importance of β-PIX to the regulation of high mobility of lung adenocarcinoma cell line H1299 and that this occurs via regulation of FA dynamics, changes in actin cytoskeleton organization and cell polarity.
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http://dx.doi.org/10.1111/jcmm.12441DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4420597PMC
May 2015

Mechanosensitive store-operated calcium entry regulates the formation of cell polarity.

J Cell Physiol 2015 09;230(9):2086-97

Department of Biomedical Engineering, National Cheng Kung University, Tainan, Taiwan.

Ca(2+) -mediated formation of cell polarity is essential for directional migration which plays an important role in physiological and pathological processes in organisms. To examine the critical role of store-operated Ca(2+) entry, which is the major form of extracellular Ca(2+) influx in non-excitable cells, in the formation of cell polarity, we employed human bone osteosarcoma U2OS cells, which exhibit distinct morphological polarity during directional migration. Our analyses showed that Ca(2+) was concentrated at the rear end of cells and that extracellular Ca(2+) influx was important for cell polarization. Inhibition of store-operated Ca(2+) entry using specific inhibitors disrupted the formation of cell polarity in a dose-dependent manner. Moreover, the channelosomal components caveolin-1, TRPC1, and Orai1 were concentrated at the rear end of polarized cells. Knockdown of TRPC1 or a TRPC inhibitor, but not knockdown of Orai1, reduced cell polarization. Furthermore, disruption of lipid rafts or overexpression of caveolin-1 contributed to the downregulation of cell polarity. On the other hand, we also found that cell polarity, store-operated Ca(2+) entry activity, and cell stiffness were markedly decreased by low substrate rigidity, which may be caused by the disorganization of actin filaments and microtubules that occurs while regulating the activity of the mechanosensitive TRPC1 channel.
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http://dx.doi.org/10.1002/jcp.24936DOI Listing
September 2015

SCP phosphatases suppress renal cell carcinoma by stabilizing PML and inhibiting mTOR/HIF signaling.

Cancer Res 2014 Dec 7;74(23):6935-46. Epub 2014 Oct 7.

Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan. Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan.

The tumor-suppressor protein promyelocytic leukemia (PML) is aberrantly degraded in multiple types of human cancers through mechanisms that are incompletely understood. Here, we show that the phosphatase SCP1 and its isoforms SCP2/3 dephosphorylate PML at S518, thereby blocking PML ubiquitination and degradation mediated by the prolyl isomerase Pin1 and the ubiquitin ligase KLHL20. Clinically, SCP1 and SCP3 are downregulated in clear cell renal cell carcinoma (ccRCC) and these events correlated with PMLS518 phosphorylation, PML turnover, and high-grade tumors. Restoring SCP1-mediated PML stabilization not only inhibited malignant features of ccRCC, including proliferation, migration, invasion, tumor growth, and tumor angiogenesis, but also suppressed the mTOR-HIF pathway. Furthermore, blocking PML degradation in ccRCC by SCP1 overexpression or Pin1 inhibition enhanced the tumor-suppressive effects of the mTOR inhibitor temsirolimus. Taken together, our results define a novel pathway of PML degradation in ccRCC that involves SCP downregulation, revealing contributions of this pathway to ccRCC progression and offering a mechanistic rationale for combination therapies that jointly target PML degradation and mTOR inhibition for ccRCC treatment.
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http://dx.doi.org/10.1158/0008-5472.CAN-14-1330DOI Listing
December 2014

GEF-H1 controls focal adhesion signaling that regulates mesenchymal stem cell lineage commitment.

J Cell Sci 2014 Oct 8;127(Pt 19):4186-200. Epub 2014 Aug 8.

Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei 11221, Taiwan

Focal adhesions (FAs) undergo maturation that culminates in size and composition changes that modulate adhesion, cytoskeleton remodeling and differentiation. Although it is well recognized that stimuli for osteogenesis of mesenchymal stem cells (MSCs) drive FA maturation, actin organization and stress fiber polarization, the extent to which FA-mediated signals regulated by the FA protein composition specifies MSC commitment remains largely unknown. Here, we demonstrate that, upon dexamethasone (osteogenic induction) treatment, guanine nucleotide exchange factor H1 (GEF-H1, also known as Rho guanine nucleotide exchange factor 2, encoded by ARHGEF2) is significantly enriched in FAs. Perturbation of GEF-H1 inhibits FA formation, anisotropic stress fiber orientation and MSC osteogenesis in an actomyosin-contractility-independent manner. To determine the role of GEF-H1 in MSC osteogenesis, we explore the GEF-H1-modulated FA proteome that reveals non-muscle myosin-II heavy chain-B (NMIIB, also known as myosin-10, encoded by MYH10) as a target of GEF-H1 in FAs. Inhibition of targeting NMIIB into FAs suppresses FA formation, stress fiber polarization, cell stiffness and osteogenic commitments in MSCs. Our data demonstrate a role for FA signaling in specifying MSC commitment.
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http://dx.doi.org/10.1242/jcs.150227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4179489PMC
October 2014

Regulation of proximal tubular cell differentiation and proliferation in primary culture by matrix stiffness and ECM components.

Am J Physiol Renal Physiol 2014 Sep 23;307(6):F695-707. Epub 2014 Jul 23.

Institute of Basic Medical Sciences, National Cheng-Kung University Medical College, Tainan, Taiwan; and Department of Physiology, National Cheng-Kung University Medical College, Tainan, Taiwan

To explore whether matrix stiffness affects cell differentiation, proliferation, and transforming growth factor (TGF)-β1-induced epithelial-mesenchymal transition (EMT) in primary cultures of mouse proximal tubular epithelial cells (mPTECs), we used a soft matrix made from monomeric collagen type I-coated polyacrylamide gel or matrigel (MG). Both kinds of soft matrix benefited primary mPTECs to retain tubular-like morphology with differentiation and growth arrest and to evade TGF-β1-induced EMT. However, the potent effect of MG on mPTEC differentiation was suppressed by glutaraldehyde-induced cross-linking and subsequently stiffening MG or by an increasing ratio of collagen in the soft mixed gel. Culture media supplemented with MG also helped mPTECs to retain tubular-like morphology and a differentiated phenotype on stiff culture dishes as soft MG did. We further found that the protein level and activity of ERK were scaled with the matrix stiffness. U-0126, a MEK inhibitor, abolished the stiff matrix-induced dedifferentiation and proliferation. These data suggest that the ERK signaling pathway plays a vital role in matrix stiffness-regulated cell growth and differentiation. Taken together, both compliant property and specific MG signals from the matrix are required for the regulation of epithelial differentiation and proliferation. This study provides a basic understanding of how physical and chemical cues derived from the extracellular matrix regulate the physiological function of proximal tubules and the pathological development of renal fibrosis.
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http://dx.doi.org/10.1152/ajprenal.00684.2013DOI Listing
September 2014