© 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016) 129, 2950-2961 doi:10.1242/jcs.183772

RESEARCH ARTICLE -11 is a novel regulator of extracellular matrix synthesis and tissue mechanics Sindhu Row1,*, Yayu Liu1,*, Stella Alimperti1, Sandeep K. Agarwal2 and Stelios T. Andreadis1,3,4,‡

ABSTRACT of mesenchymal stem cells (MSCs) into contractile smooth muscle We discovered that Cadherin-11 (CDH11) regulates collagen and cells (SMCs) by regulating expression of the key transcription factor elastin synthesis, both affecting the mechanical properties and SRF. In addition, CDH11 is necessary for development of contractile function of animal tissues. Using a Cdh11-null mouse contractility of smooth-muscle-containing tissues such as artery model, we observed a significant reduction in the mechanical properties and bladder (Alimperti et al., 2014). [Youngs’ modulus and ultimate tensile strength (UTS)] of Cdh11−/− as Extracellular matrix such as collagens and elastin are compared to wild-type (WT) mouse tissues, such as the aorta, bladder abundant in all body tissues and play crucial roles in development, and skin. The deterioration of mechanical properties (Youngs' modulus tissue remodeling following injury, and maintenance of tissue and UTS) was accompanied by reduced collagen and elastin content in mechanics and function in homeostasis. As a result, loss of collagen Cdh11−/− mouse tissues as well as in cells in culture. Similarly, knocking and/or elastin has been implicated in many diseases and is well- down CDH11 abolished collagen and elastin synthesis in human cells, documented in aging (Kohl et al., 2011; Wagenseil and Mecham, and consequently reduced their ability to generate force. Conversely, 2012). For example, fragmented or irregular distribution of collagen engagement of CDH11 through homophilic interactions, led to swift and elastin fibrils have been implicated in bladder incontinence as activation of the TGF-β and ROCK pathways as evidenced by well as cardiovascular disorders such as hypertension, aneurysms ’ phosphorylation of downstream effectors. Subsequently, activation of and Marfan s syndrome (Goepel and Thomssen, 2006; Li, 2012a; the key transcription factors, MRTF-A (also known as MKL1) and Benke et al., 2013). By contrast, excessive and cumulative MYOCD led to significant upregulation of collagen and elastin . deposition of collagen disrupts organ architecture, leading to scar Taken together, our results demonstrate a novel role of adherens formation and loss of function. Recent studies have implicated junctions in regulating extracellular matrix (ECM) synthesis with CDH11 in lung and skin fibrosis (Schneider et al., 2012; Agarwal, implications for many important biological processes, including 2014; Wu et al., 2014), myofibroblast migration to site of lung maintenance of tissue integrity, wound healing and tissue regeneration. fibrosis (Schneider et al., 2012) and mesenchymal stem cell differentiation into smooth muscle cells (Alimperti et al., 2014). KEY WORDS: Cadherin-11, Extracellular matrix, However, it is not clear whether CDH11-mediated adherens Tissue regeneration, MRTF-A, Myocardin, Collagen, Elastin, junctions regulate ECM production and the signaling mechanisms Mechanical properties that might be involved remain unknown. To this end, we set out to determine the role of CDH11 in ECM INTRODUCTION production and identify the mechanism of action. Our work was Cell– through plays an important role in motivated by our initial discovery that smooth-muscle- or multiple aspects of cellular behavior including proliferation, myofibroblast-containing tissues such as aorta, bladder and skin differentiation, apoptosis, cell polarity (Niessen and Gumbiner, from CDH11-null mice (Cdh11−/−) exhibited significantly impaired 2002; Cavallaro and Dejana, 2011), embryonic stem cell self- mechanical strength as well as significantly reduced collagen and renewal and differentiation (Li et al., 2012b), tissue morphogenesis elastin content, as compared to WT mice. This is not only a novel but and maintenance of tissue integrity (Harris and Tepass, 2010). also very surprising result given that Cdh11−/− mice develop CDH11 is expressed in osteoblasts and mesenchymal cells as well normally, are fertile and display no obvious phenotype other than as in epithelial cells undergoing epithelial–mesenchymal transition modest osteopenia (Shin et al., 2000; Kawaguchi et al., 2001a,b) and (EMT), as for example during progression of cancer cells into a decreased lung and skin fibrosis after lung injury (Schneider et al., metastatic state (Kimura et al., 1995; Tomita et al., 2000; Zeisberg 2012; Wu et al., 2014). Using a combination of knockdown and Neilson, 2009). Recent work from our laboratory has shown and gain-of-function approaches, we discovered a direct and novel that cell–cell adhesion through CDH11 is crucial for differentiation role of molecule CDH11 in extracellular matrix (ECM) production and identified the pathways and transcription factors mediating this action. Taken together, our results implicate 1Department of Chemical and Biological Engineering, University at Buffalo, cell–cell adhesion as a regulator of ECM production and suggest State University of New York, Amherst, NY 14260, USA. 2Section of Allergy, Immunology, and Rheumatology Biology, Inflammation Center, Baylor College of new ways through which adherens junctions might regulate Medicine, Houston, TX 77030, USA. 3Department of Biomedical Engineering, important biological processes from maintaining tissue integrity to University at Buffalo, State University of New York, Amherst, NY 14260, USA. promoting wound healing and tissue regeneration. 4Center of Excellence in Bioinformatics and Life Sciences, Buffalo, NY 14203, USA. *These authors contributed equally to this work RESULTS ‡ Author for correspondence ([email protected]) Cdh11−/− mouse tissues exhibit diminished mechanical ’ S.T.A., 0000-0001-9885-0457 properties such as Youngs modulus and UTS Recent work from our laboratory demonstrated that loss of CDH11

Received 24 November 2015; Accepted 10 June 2016 impaired the myogenic differentiation capacity of MSCs and Journal of Cell Science

2950 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 2950-2961 doi:10.1242/jcs.183772 diminished the contractile ability of SMCs that were derived from (Fig. 1C). Interestingly, the collagen layer (blue) in the muscle layer of MSCs. In addition, SMC-containing tissues, such as the aorta and aorta and bladder of Cdh11−/− animals also appeared thinner (see also bladder, exhibited significantly reduced levels of SMC proteins and Table S1). In addition, quantitative measurements of collagen content diminished contractile ability compared with WT controls using the hydroxyl-proline assay, showed a significant 70% reduction (Alimperti et al., 2014). These results prompted us to hypothesize (n=4, P<0.05, unpaired two-tailed Student's t-test) in the Cdh11−/− that loss of CDH11 might also affect the mechanical properties of bladder and aorta tissues (Fig. 1D). Values were internally normalized these tissues or organs, possibly by affecting expression of to WT tissues after the collagen content of each tissue specimen was extracellular matrix components. normalized to its own dry weight. Therefore, although the absolute To address this hypothesis, first we examined the mechanical value of collagen was also lower for Cdh11−/− skin, the dry weight of properties of smooth-muscle- and myofibroblast-containing tissues, WT skin of equal surface area was higher, thereby bringing the such as aorta, bladder and skin of 6–8-week-old Cdh11−/− and WT normalized collagen content closer to that of Cdh11−/− tissues. mice. Tissue rings were mounted onto an Instron tensile tester and Similarly, Verhoeff’s staining showed fewer elastin fibers in stretched unilaterally with constant strain until the yielding point. Cdh11−/− tissues (Fig. 1E, brown-black fibers), as evidenced by The ultimate tensile stress (UTS) and Young’s modulus were lower staining intensity and lack of elastin fiber continuity (see normalized to corresponding values of WT tissues (Fig. 1A,B). higher magnification images in Fig. S1A). Quantitative Surprisingly, we observed a significant reduction in both the UTS measurement of elastin using the colorimetric ninhydrin assay and Young’s modulus of Cdh11−/− mice tissues (40–60%, n=4, revealed that Cdh11−/− tissues contained 30–60% (P<0.05, n=9, P<0.05), indicating that loss of CDH11 correlated with impaired unpaired two-tailed Student's t-test) less fibrous elastin than their mechanical strength. WT counterparts (Fig. 1F). Taken together, the reduction in collagen and elastin content is consistent with the reduced −/− Cdh11−/− mouse tissues exhibit impaired ECM production mechanical properties exhibited by the Cdh11 tissues. Given that the mechanical properties are derived mostly from extracellular matrix (ECM) we investigated the collagen and elastin Cdh11−/− cells exhibit reduced expression of collagen and −/− content of aorta, bladder and skin from Cdh11 and WT mice. elastin genes Masson’s trichrome staining (blue) revealed a reduced amount of Next, we compared the capacity of Cdh11−/− versus WT cells to collagen in all three tissues of Cdh11−/− as compared to WT mice synthesize ECM. To this end, dermal fibroblasts and aortic smooth

−/− Fig. 1. Mechanical properties and ECM composition of Cdh11 and WT mouse tissues. (A) Ultimate tensile strength (UTS) and (B) Young’s moduli of bladder, skin and aorta tissues reported as normalized to corresponding WT tissues (n=4). (C) Histological examination of collagen content in cross-sections of − − skin, aorta and bladder tissue from Cdh11 / and WT mice by Masson’s trichrome staining. Scale bars: 200 µm. (D) Collagen content as quantified by hydroxyl- − − proline assay in Cdh11 / skin, aorta and bladder tissues and reported after normalization to WT (n=4). (E) Histological examination of elastin content in cross- − − sections of skin, aorta and bladder tissue from Cdh11 / and WT mice by Verhoeff’s elastin staining. Scale bars: 200 µm. (F) Elastin content as quantified by − − ninhydrin assay in Cdh11 / skin, aorta and bladder tissues and reported as after normalization to WT (n=4). All quantitative results are mean±s.d. *P<0.05 as compared to WT (unpaired two-tailed Student’s t-test). Journal of Cell Science

2951 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 2950-2961 doi:10.1242/jcs.183772 muscle cells (ASMCs) from WT and Cdh11−/− mice were tested compromised as compared to their WT counterparts. Specifically, for expression levels of ECM genes (Fig. 2A). Interestingly, shCDH11 cells exhibited considerably lower levels of COL1A1 quantitative real-time PCR (qRT-PCR) revealed that Cdh11−/− (∼120-fold), COL3A1 (∼480-fold) and ELN (∼11-fold) compared ASMCs exhibited significant loss in collagen and elastin mRNA to WT human dermal fibroblasts (Fig. 3A). The levels of (∼10-fold for Col1a1 and Eln,20–30-fold for Col1a2 and Col3a1), CDH11, ELN, COL1α2 and COL3α1 were also reduced but the loss was more dramatic for Cdh11−/− dermal fibroblasts dramatically as assessed by western blotting (Fig. 3B) and (∼450-fold for Col1a1, ∼90-fold for Col1a2, ∼1200-fold for immunostaining for COL3α1 and ELN (Fig. 3C). Moreover, Col3a1 and ∼12-fold for Eln). Similar results were observed at knocking down of CDH11 significantly reduced the mRNA and the protein level as evidenced by western blots showing protein levels of transcription factors that have been implicated in significantly decreased protein levels of COL1α2, COL3α1 and collagen production such as MYOCD, SRF, and MRTF-A and -B, to a lesser extent ELN in Cdh11−/− cells (Fig. 2B). This result was in agreement with our findings for mouse dermal fibroblasts further confirmed by immunohistochemistry for COL1 and ELN (Fig. S2A,B). (Fig. 2C,D). In agreement, the mRNA levels of several In addition, transcriptional activity was measured through the transcription factors that are known to regulate collagen and activity of the CArG response element (CArG-RE) or serum elastin expression such as Myocd, Mrtfa (also known as Mkl1), response element (SRE), using a triple promoter lentiviral vector Mrtfb (also known as Mkl2) and Srf were significantly reduced in that was previously developed in our laboratory (Alimperti et al., Cdh11−/− as compared to WT mouse dermal fibroblasts (Fig. S1B). 2012). This vector encodes for ZsGreen under the CArG-RE, By contrast, the level of Fbn1 remained unaffected (Fig. S1C). DsRed2 under the constitutive human PGK promoter and shRNA These results indicate a strong relationship between CDH11 and under a tetracycline regulatable H1 promoter in the viral LTR. ECM production. Transduced cells are expected to express DsRed constitutively but express ZsGreen only upon CArG-RE activation, which can be Cdh11−/− dermal fibroblasts displayed reduced contractility quantified by fluorescence microscopy. As shown in Fig. S2C, and ECM production in 3D knocking down CDH11 reduced the CArG-RE activity significantly The above results prompted us to examine whether loss of Cdh11 as compared to control cells (scrambled shRNA), suggesting might affect the ability of myofibroblasts to generate force. To this reduced SRF transcriptional activity. end, cells were embedded in fibrin hydrogels (106 cells/ml) and 1 h The contractility of shCDH11 cells was also tested using 3D after polymerization the gels were released from the plate wall and fibrin gels, as described above. As shown in Fig. 3D, fibrin gel allowed to undergo compaction in the presence of TGF-β1. After compaction was abolished upon CDH11 knockdown. The collagen 48 h, the area of each gel was measured and normalized to its initial content of fibrin hydrogels containing shCDH11 human dermal area. As shown in Fig. 2E, Cdh11−/− cells exhibited significantly fibroblasts was reduced by ∼50% as compared to hydrogels impaired ability to show compaction as compared to WT cells containing control cells (Fig. 3E). Finally, reduced collagen (Cdh11−/−, 43±6% versus WT, 76±9% of the initial gel area, n=6, production was accompanied by severely compromised P<0.05; mean±s.d.). Furthermore, the initial rate of compaction was mechanical strength as evidenced by a reduction in the UTS ∼2.38% per hour for WT cells but less than half of that at ∼1.01% (∼70%, n=4, P<0.05) and Young’s modulus (∼40%, n=4, P<0.05) per hour for Cdh11−/− cells. of tissue constructs containing shCDH11 human dermal fibroblasts We also measured the mechanical properties and collagen (Fig. 3F,G). content of tissue constructs prepared from Cdh11−/− or WT mouse dermal fibroblasts, as we described in previous publications (Diaz- CDH11 engagement induces ECM synthesis through the TGF- Chavez et al., 2008; Liang et al., 2013; Koobatian et al., 2016). To β and ROCK pathways this end, we prepared cylindrical tissue equivalents by embedding Cell–cell-contact-induced signaling follows engagement of cells in fibrin hydrogels that were polymerized around cylindrical cadherins on the surface of a cell in homotypic interactions with mandrels and cultured in the presence of TGF-β1 for 2 weeks. At cadherins on the surface of a neighboring cell. However, formation that time, the collagen content was measured using the hydroxyl- of adherens junctions is also followed by other events such as proline assay and showed that Cdh11−/− dermal fibroblasts formation of gap junctions. To isolate the effects of CDH11 produced only 28.3±0.69% (n=6, P<0.05) of the collagen signaling from cadherin-dependent juxtacrine signals, we employed amount produced by WT cells (Fig. 2F). In addition, both the a fusion protein of CDH11 with the human Fc region of IgG, herein UTS and Young’s modulus of Cdh11−/− tissue constructs were indicated as CDH11-Fc, which was used to coat the surface of non- significantly lower at ∼55% (n=6, P<0.05) of tissue constructs tissue culture plates before addition of cells to initiate cadherin containing WT cells (Fig. 2G,H). These results show that, engagement (Fig. 4A). consistent with the in vivo data, Cdh11−/− dermal fibroblasts Interestingly, after 2 days on CDH11-Fc, the mRNA levels of exhibited significantly impaired capacity to generate force and COL1A1, COL1A2, COL3A1 and ELN were significantly higher remodel 3D tissue constructs, which exhibited compromised [by 5–10-fold for collagen genes and 20-fold for the elastin gene mechanical properties. as compared to cells on tissue culture (TC) surface] (Fig. 4B). Increasing the surface concentration of CDH11-Fc led to a step-wise Knockdown of CDH11 in human dermal fibroblasts increase in CDH11, as well as of COL1α2 and ELN protein levels compromises ECM production (Fig. S2D), supporting the notion that the observed effects were Next, we tested whether the relationship between loss of CDH11 due to increased number of CDH11 contacts. In addition, and ECM deposition holds for human cells as well, by knocking overexpression of CDH11 using lentiviral delivery (CDH11+ down CDH11 in human neonatal foreskin dermal fibroblasts using cells) increased expression of COL1α2 and COL3α1 proteins short hairpin RNA (shRNA)-encoding lentivirus (shCDH11). In significantly only when cells were plated on CDH11-Fc surface agreement with Cdh11−/− mouse cells, the ability of shCDH11 (Fig. S2E), suggesting that engagement of CDH11 in homotypic human dermal fibroblasts to synthesize ECM was severely contacts was necessary for ECM production. Journal of Cell Science

2952 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 2950-2961 doi:10.1242/jcs.183772

−/− Fig. 2. ECM synthesis in Cdh11 and WT mouse cells. (A) qRT-PCR for the indicated genes. The relative mRNA levels are reported as the fold change with − − respect to Cdh11 / samples (n=4). Dermal fibroblasts (DFs) are shown in top panels (solid) and ASMCs are shown in bottom panels (hatched). (B) Protein levels − − of COL1α2, ELN and COL3α1 as analyzed by western blotting. Quantification of the band intensity for each protein in Cdh11 / cells normalized to the corresponding band in WT cells (n=3). (C) Immunostaining for CDH11, COL1 and ELN (green) and counterstained with DAPI (blue). Scale bar: 100 µm. − − (D) Normalized intensity per cell quantified from n=5 fields of view. (E) Compaction of fibrin hydrogels containing Cdh11 / or WT cells. Upper panel, picture of hydrogels around a cylindrical mandrel after 48 h. The dotted lines demarcate the inside and outside diameter for each gel. Lower panel, average of the area of each gel as a fraction of the initial gel area (A/A0) plotted over time (h) (n=6). (F) Collagen content of fibrin gel rings as quantified by hydroxyl-proline assay and − − normalized to WT (n=6). (G) Ultimate tensile strength (UTS) and (H) Young’s modulus of fibrin gel rings containing Cdh11 / or WT cells. The data are normalized

to the corresponding WT rings (n=6). All quantitative results are mean±s.d. *P<0.05 as compared to WT (unpaired two-tailed Student's t-test). Journal of Cell Science

2953 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 2950-2961 doi:10.1242/jcs.183772

Fig. 3. ECM synthesis in shCDH11 and control human dermal fibroblasts. (A) The relative mRNA levels are reported as the fold change with respect to shCDH11 samples (n=4). *P<0.05 as compared to shCDH11 cells. (B) Protein levels of CDH11, ELN, COL1α2, and COL3α1 as analyzed by western blot. GAPDH served as a loading control. A quantification is also shown (n=4). *P<0.05 as compared to control. (C) Immunostaining for COL3 and ELN (green) and counterstained with DAPI (blue). Scale bar: 100 µm. A quantification is also shown (n=4). *P<0.05 as compared to control. (D) Compaction of fibrin hydrogels containing shCDH11 or control human dermal fibroblasts. Upper panel, picture of hydrogels around a cylindrical mandrel after 48 h. The dotted lines demarcate the inside and outside diameter for each gel. Lower panel, average of the area of each gel as a fraction of the initial gel area (A/A0) plotted over time (h) (n=6). (E) Collagen content of fibrin gel rings as quantified by hydroxyl-proline assay and normalized to WT (n=6). *P<0.05 as compared to control. (F) Ultimate tensile strength (UTS) and (G) Young’s modulus of fibrin gel rings containing shCDH11 or WT cells. The data are normalized to the corresponding control rings (n=6). *P<0.05 as compared to control. All quantitative results are mean±s.d. and P-values were calculated with an unpaired two-tailed Student's t-test.

Given that the TGF-β and ROCK pathways are well known for phosphorylated (p-)SMAD2, indicating that the phosphorylation regulating ECM synthesis, the contribution of these pathways to of SMAD2 was induced by the CDH11-Fc surface, independent of CDH11-mediated signaling was investigated with the aid of the TGF-β-ligand–receptor interactions (Fig. 4D; Fig. S3A). By CDH11-Fc surface. Surprisingly, plating cells on CDH11-Fc contrast, engagement of N-cadherin (CDH2) on CDH2-Fc surface induced phosphorylation of SMAD2 and MYPT within 2 h, led to much less phosphorylation of MYPT and its effect suggesting that engagement of CDH11 was sufficient to activate on SMAD2 was negligible as compared to that of CDH11-Fc the TGF-β and ROCK pathways (Fig. 4C). Indeed, blocking TGF-β (Fig. S3B), suggesting that CDH11 is the dominant cadherin by using a neutralizing antibody had no effect on the level of activating these two pathways. Journal of Cell Science

2954 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 2950-2961 doi:10.1242/jcs.183772

Fig. 4. CDH11-Fc induced ECM production is mediated through the TGF-β and Rho/ROCK signaling. (A) Schematic of CDH11-Fc coating. (B) The relative mRNA levels are reported as the fold change with respect to TC samples (n=4). *P<0.05 as compared to on tissue culture plates (TC). (C) Activation of ROCK and TGF-β pathways on CDH11-Fc vs tissue culture plates as indicated by MYPT and SMAD-2 phosphorylation (P-MYPT and P-SMAD2, respectively). The cells were plated on CDH11-Fc-coated or tissue culture plates in the presence or absence of SB43 or Y27, chemical inhibitors of ROCK or TGF-β pathways, respectively. After 2 h, the cells were lysed and the levels of phosphorylated MYPT or SMAD2 were measured by western blotting. (D) Protein level of P-SMAD2 upon addition of TGF-β-neutralizing antibody (AbTGF-β). Quantification is shown as relative levels of P-SMAD2 relative to SMAD2. Values reported are normalized to housekeeping gene GAPDH (n=3). *P<0.05 as compared to on tissue culture plates. (E) qRT-PCR for the indicated genes. The relative mRNA levels are reported as the fold change with respect to tissue culture plate samples (n=4). *P<0.05 as compared to on tissue culture plates; #P<0.05 as compared to CDH11-Fc. (F) Protein levels of COL1α2, COL3α1 and ELN as measured by western blotting. GAPDH served as a loading control. (G) Quantification of the band intensity for each protein on CDH11-Fc in the presence or absence of SB43 or Y27 cells normalized to the corresponding band on TC (n=4). *P<0.05 as compared to on tissue culture plates; #P<0.05 as compared to CDH11-Fc. (H) Immunohistochemistry images for COL1, COL3 or ELN (green), counterstained with DAPI (blue). Scale bars: 50 µm. All quantifications results are mean±s.d. and P-values were calculated with an unpaired two-tailed Student’s t-test.

In addition, blocking the TGF-β1 pathway by use of SB43152 CDH11-induced collagen and elastin synthesis is mediated (SB43) or the ROCK pathway by use of Y27632 (Y27) reduced by MRTF and MYOCD significantly the CDH11-Fc-mediated increase of COL1A1, To further investigate the targets of the CDH11 signaling, several COL1A2, COL3A1 and ELN mRNA (Fig. 4E). Similarly, both key transcription factors that are known to mediate collagen and inhibitors decreased COL1α2, COL3α1 and ELN proteins as elastin transcription, such as SRF, MYOCD and MRTF-A and -B shown by western blotting (Fig. 4F,G) and immunostaining were assessed by qRT-PCR (Fig. 5A). Interestingly, cell adhesion (Fig. 4H), although the TGF-β1 pathway seemed to have a greater on the CDH11-Fc surface significantly increased the mRNA levels effect, especially on ELN expression. Interestingly, blocking the of CDH11 itself (∼25-fold, n=3, P<0.05), as well as SRF (∼6-fold, ROCK pathway with Y27 had little or no effect on the n=3, P<0.01), MRTF-A (11-fold, n=3, P<0.05), MRTF-B (9-fold, phosphorylation of SMAD2, whereas blocking the TGF-β n=3, P<0.05) and especially MYOCD (50-fold, n=3, P<0.05). This pathway with SB43 reduced p-MYPT levels as well (Fig. 4C). increase was mediated through the ROCK pathway, as the mRNA Similarly, the RhoA inhibitor, C3 eliminated p-MYPT but had no levels of CDH11 and all transcription factors were significantly effect on p-SMAD2 (Fig. S3C). These results indicate partial reduced by Y27 (SRF, MRTF-A and -B were reduced by 3–5-fold, activation of the ROCK pathway by TGF-β1, which could also n=3, P<0.05). By contrast, blocking the TGF-β1 pathway had no explain the greater decline of ECM synthesis upon treatment with effect, except on MYOCD, which decreased significantly by ∼5-

SB43. fold (n=3, P<0.05). In addition, blocking RhoA with C3 also Journal of Cell Science

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Fig. 5. CDH11-Fc engagement induces expression of several transcription factors. (A) Cells were plated on CDH11-Fc or tissue culture plates (TC) in the presence or absence of SB43 or Y27 and the next day (16 h later) they were lysed and the levels of indicated genes were measured by qRT-PCR. The relative mRNA levels are reported as the fold change with respect to tissue culture plate samples (n=4). *P<0.05 as compared to tissue culture plate; #P<0.05 as compared to CDH11-Fc. (B) Immunostaining for MRTF-A (green) on CDH11-Fc in the presence or absence of SB43 or Y27; cells were counterstained with DAPI. Cells on tissue culture plates were used as a control. Scale bars: 50 µm. A quantification of the relative nuclear signal of MRTF-A shown in right panel (n=5 fields of view). *P<0.05 as compared to tissue culture plates. All quantifications results are mean±s.d. and P-values were calculated with an unpaired two-tailed Student’s t-test. eliminated the CDH11-mediated increase in of shown in Fig. S4C, lack of MRTF-A or MYOCD impaired collagen MRTF-A and -B, MYOCD and SRF (Fig. S3D), implicating the and elastin production, even in the presence of CDH11, suggesting canonical Rho–ROCK pathway in MRTF-A and -B transcription. that both transcription factors are necessary for CDH11-induced Notably, adhesion to CDH11-Fc significantly increased the protein ECM synthesis. level of MRTF-A in the cell nucleus, which was blocked by Y27 but was unaffected by SB43 (Fig. 5B). Conversely, knocking down DISCUSSION CDH11 with shRNA reduced expression and completely blocked We have previously shown that in high-density MSC cultures, MRTF-A nuclear localization (Fig. S4A). CDH11-mediated intercellular adhesion activated ROCK leading to mesenchymal stem cell differentiation into smooth muscle Loss of MRTF-A, MRTF-B or MYOCD abolishes CDH11-Fc- cells (Alimperti et al., 2014). In this study, we presented in vivo and mediated collagen and elastin production in vitro experimental evidence that CDH11 is necessary for To determine which transcription factor(s) mediated CDH11-Fc- extracellular matrix production contributing to the mechanical induced collagen and elastin production, we employed shRNA- properties of tissues. We discovered that smooth-muscle-containing encoding lentivirus to knockdown MYOCD, MRTF-A and -B or tissues of Cdh11−/− mice exhibited significantly reduced SRF (shMYOCD, shMRTF-A/B and shSRF, respectively). mechanical strength, which correlated with significant reduction Knocking down MRTF-A and -B, or MYOCD drastically reduced in collagen and elastin content. Indeed, expression of Col1a1, the mRNA levels of COL1A1, COL3A1 and ELN (Fig. 6A,B) Col1a2, Col3a1 and Eln at the mRNA as well as the protein level as well as the protein levels of COL1A2, COL3A1 and ELN were dramatically reduced both in mouse Cdh11−/− fibroblasts and (Fig. 6C,D), indicating that both transcription factors were required human shCDH11 fibroblasts. Similarly, mRNAs of key for transciptional activation of collagen and elastin genes. By transcription factors that are known to regulate collagen and contrast, knocking down SRF affected only CDH11-Fc mediated elastin were reduced so as to be effectively not present. In addition, ELN production but had no significant effect on COL1α2or the force generation ability of cells lacking CDH11 was COL3α1 (Fig. S4B). dramatically reduced as shown by their ability to compact fibrin Interestingly, CDH11 levels were reduced dramatically by loss of hydrogels. These data implicate intercellular adhesion as a new and MRTF-A and -B (∼50-fold, n=3, P<0.03) and to a much lesser crucial regulator of ECM production with significant consequences extent by loss of MYOCD (∼3-fold, n=3, P<0.03) (Fig. 6A,B). In for tissue biomechanics. order to determine whether loss of MRTF-A or MYOCD affected Using a CDH11-Fc-decorated surface, we identified the ECM synthesis directly or indirectly by decreasing CDH11, we signaling pathways mediating CDH11-induced ECM production. measured the protein levels of COL1A2, COL3A1 and ELN in Examination of early signaling dynamics revealed activation of both shMRTF-A/B or shMYOCD cells overexpressing CDH11. As the ROCK and TGF-β pathways shorty after cell attachment to the Journal of Cell Science

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Fig. 6. CDH11-inducedECM synthesis ismediated byMRTF-Aand MYOCD. qRT-PCR datafor CDH11, MRTF-A, MYOCD, COL1A1, COL1A2, COL3A1 and ELN for (A) shMRTF-A and (B) shMYOCD human dermal fibroblasts. The relative mRNA levels are reported as the fold change with respect to tissue culture platesamples (n=4). *P<0.05 as compared to the indicated knockdown sample (unpaired two-tailed Student’s t-test). Protein levels of MYOCD, MRTF-A, ELN, COL1α2, COL3α1as analyzed by western blot in (C) shMRTF-A or (D) shMYOCD human dermal fibroblasts and control dermal fibroblasts. All quantitative results are mean±s.d. surface (within 2 h or less), as evidenced by phosphorylation of expression of transcription factors known to affect ECM synthesis MYPT and SMAD2, respectively. Interestingly, blocking TGF-β1 (Ueyama et al., 2003; Medjkane et al., 2009; Small et al., 2010; with a function-blocking antibody had no effect on CDH11- Leitner et al., 2011; Velasquez et al., 2013; Johnson et al., 2014; mediated SMAD2 phosphorylation, suggesting that activation of Parreno et al., 2014), such as MYOCD, MRTF-A, SRF and to a the TGF-β pathway was the direct effect of CDH11 engagement, lesser extent MRTF-B. In agreement, knocking down either rather than through paracrine action of de novo transcribed soluble MYOCD or MRTF-A and -B decreased CDH11-mediated TGF-β1. This result suggests that engagement of CDH11 might expression of collagen and elastin, but the effects of MRTF-A activate the TGF-β receptor or phosphorylate SMAD2 or SMAD3 and -B were significantly stronger. Although past studies have only through a currently unidentified pathway. Interestingly, CDH2 recognized MRTF and MYOCD as co-activators of SRF, some engagement activated ROCK to a much lesser extent and failed recent studies have shown that they can act as transcription factors to phosphorylate SMAD2, indicating a unique role of CDH11 in independently (Tang et al., 2008; Asparuhova et al., 2011; activating the TGF-β1 pathway. Luchsinger et al., 2011; Smith et al., 2012; Kitchen et al., 2013). In addition, activation of the TGF-β pathway by CDH11 had a MRTF-A has been shown to control Col1A2 promoter activity as direct effect on ROCK activation, as inhibition of SMAD2 well as expression of other extracellular matrix molecules or SMAD3 phosphorylation also reduced the phosphorylation of (Asparuhova et al., 2011; Luchsinger et al., 2011). MYOCD has MYPT. The converse was not true, as inhibition of ROCK also been implicated in studies showing CArG-RE-independent or RhoA had no effect on CDH11-induced phosphorylation of regulation of ECM (Tang et al., 2008; Kitchen et al., 2013). SMAD2 or SMAD3. The crosstalk between the two pathways has Interestingly, engagement of CDH11 on surface-immobilized been previously reported especially in the context of the role of CDH11-Fc increased the mRNA levels of all these transcription TGF-β in ECM production (Itoh et al., 2007; Zhu et al., 2013) but factors significantly. In agreement, the CArG-RE activity was our study demonstrates that such crosstalk also exists in the blocked almost completely when CDH11 was knocked down by context of CDH11-mediated intercellular adhesion. Currently, the shRNA. CDH-11-mediated MRTF-A and -B expression was nature of the effector(s) mediating this inter-connection remains dependent solely on ROCK, whereas MYOCD required both unknown. ROCK and TGF-β1 activation. Given that expression of the ECM Furthermore, loss of CDH11 either in Cdh11−/− mouse dermal genes COL1A1, COL1A2, COL3A1 and ELN depended on both fibroblasts or in shCDH11 human dermal fibroblasts abolished the pathways, we conclude that TGF-β is required to promote Journal of Cell Science

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Fig. 7. Schematic showing proposed mechanism of ECM synthesis following CDH11 engagement. CDH11 engagement activates TGF-β and ROCK pathways as evidenced by phosphorylation of MYPT and Smad2. Activation of Rho–ROCK leads to polymerization and nuclear localization of MRTF-A. Phosphorylation of Smad2–Smad3 leads to increased levels of MYOCD. These two transcription factors, perhaps with the help of other co-factors such as SRF, bind to the SBE or CARG response elements leading to transcription of collagen and elastin as well as CDH11, indicating the presence of a positive-feedback loop. The dotted lines denote that the pathway is not fully defined. expression of MYOCD, whereas ROCK activation is necessary for and skin, among other tissues (Kohl et al., 2011; Wagenseil and expression of MRTF-A and -B (see Fig. 7). Interestingly, activation Mecham, 2012). In this context, strategies that promote CDH11 of the Rho–ROCK pathway by CDH11 engagement led to increased engagement might be a viable strategy to restore the levels of ECM, MRTF-A and -B, MYOCD and SRF gene expression, ultimately thereby preventing deterioration of the mechanical properties and leading to high levels of ECM synthesis. the proper physiological function of these tissues. Our results might have implications for restoration of tissue Interestingly, engagement of CDH11 increased the expression function, wound healing and regenerative medicine. Recent studies of CDH11 itself, thereby suggesting the presence of a positive- implicated CDH11 in lung and skin fibrosis following chemical feedback loop, which required the presence of MRTF-A- and injury, leading to impaired wound healing and loss of function -B but not MYOCD, as shown by knockdown experiments. (Schneider et al., 2012; Wu et al., 2014). Our results provide Overexpression of CDH11 did not increase ECM production mechanistic insight into this process and suggest, for the first time, until the cells were plated either at high density or on surface- that CDH11 might be contributing to collagen deposition following immobilized CDH11-Fc, suggesting that CDH11 engagement in injury, by directly activating both the TGF-β and ROCK pathways, homophilic interactions was necessary for the activation of signaling with subsequent activation of the transcription factors necessary for pathways leading to expression of CDH11, collagen type I and III and ECM production. Interestingly, CDH2 engagement did not have the elastin. Interestingly, formation of adherens junctions through same effects, suggesting that CDH11 might be a distinct target for cadherins has been shown to generate mechanical forces between preventing fibrosis and promoting tissue regeneration. By contrast, a adjacent cells (Tzima et al., 2005; Liu et al., 2007b), ultimately number of diseases are attributed to a loss or impaired state of the regulating basic cellular functions such as proliferation, ECM, for example, stress urinary incontinence or improper bladder differentiation and migration. Given that engagement of CDH11 function, which is related to irregular distribution of elastin fibers induced ECM production, our findings implicate intercellular (Goepel and Thomssen, 2006) and reduced collagen type I and III adhesion force as a new and crucial regulator of ECM production, (Li et al., 2012a), and Marfan’s syndrome, which is caused by a ultimately affecting the mechanical properties of tissues and mutation in the fibrillin gene leading to lack of elastin fibers and consequently biological processes such as tissue development, reduced tissue levels of TGF-β in the aorta, with subsequent regeneration and wound healing. Our findings reveal new functions increase in stiffness (Benke et al., 2013). However, loss of CDH11 of CDH11 with significant repercussions for the function of vital had no effect on the mRNA expression of the fibrillin gene, as tissues including the vasculature. It would be interesting to determine shown in Cdh11−/− mouse dermal fibroblasts. In addition, age, whether these observations can be extended to other smooth-muscle- hypertension and are associated with containing tissues or organs such as the lung, stomach and intestine, significantly reduced collagen and elastin content in large arteries as well as to other muscle types such as skeletal or . Journal of Cell Science

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MATERIALS AND METHODS amino acids (Hyclone Labs, GE Healthcare, Logan, UT), supplemented with Animals and tissue collection 2 ng/ml bFGF. These cells were used between passage 2 and 6. Cdh11−/− mice and wild-type B6:129 F1 intercross mice were bred and maintained as previously described (Schneider et al., 2012), and were Knockdown of genes by RNA interference housed at Baylor College of Medicine with the approval of the Baylor shLVDP, as characterized by our group in a previous study (Alimperti et al., College of Medicine Institutional Animal Care and Use Committee. Mice 2012) was utilized and modified by cloning in the oligonucleotide were killed at 6–8 weeks of age in a standard CO2 chamber, and tissues, sequences presented in Table S2, using ClaI and MluI restriction enzyme namely, aorta, bladder and skin (from the back) were immediately sites in the 3′ LTR of the vector. Resulting plasmids were used along with harvested. Fresh tissue was reserved for mechanical testing after extensive psPAX2 and PMD2.G in a standard calcium phosphate transfection method washes in PBS. Tissues used for histological analyses were fixed in 10% using HEK 293T to make lentivirus as described elsewhere (Alimperti et al., neutral buffered formalin. 2012). Lentiviral transduction of human dermal fibroblasts was carried out with 8 µg/ml polybrene, and infected cells were immediately used for Mechanical testing of tissues experimental analyses. Tissues obtained from mice were cut into rings (for aorta and bladder) or strips (for skin tissue) and diameters or thicknesses were measured for Overexpression of CDH11 calculation of contact area. These were subjected to mechanical testing using The CDH11-encoding region was PCR-amplified from pCMV-sport6- a uniaxial Instron tensile tester (Model 3343, 50N load cell, Instron OBCAD (Open Biosystems) using PCR primers (5′-AACAAACCGGTT- Corporation, Norwood, MA) as previously described (Row et al., 2015). TGGGCCCCTCGAGGGATA-3′ sense and 5′-ACAAGCTAGCGGTCC- Ring samples or strips were mounted onto tester grips directly (for strips of GGAATTCCCGGGATA-3′ antisense) and ligated into pSIN-EF2-Sox2- skin) or by using stainless steel hooks (for aorta and bladder tissue rings). puro (Addgene) using restriction enzyme BamHI. The resulting vector was Samples were displaced in the vertical axis at constant speed (18 mm/min named pSIN-EF2-OBCAD-puro, which was used for lentiviral delivery crosshead speed) until failure. Young’s moduli were obtained from the after packaging in HEK 293T cell line as described above. linear portion of the stress–strain curve, exported from the software (Bluehill 3, Instron Corporation, Norwood, MA) and the UTS was calculated as the CDH11-Fc- and CDH2-Fc-coated surface breaking force per unit area of tissue and expressed in MPa. Non-tissue culture plates were utilized owing to their hydrophobicity, and coated with goat anti-human IgG (FCγ-specific, Jackson ImmunoResearch, Colorimetric quantification of collagen and elastin in tissues West Grove, PA) in attachment buffer overnight at 4°C as described A ninhydrin assay was used for evaluation of elastin content and a hydroxyl- previously (Lira et al., 2008). 50 ng/cm2 of recombinant human CDH11-Fc proline assay was used for the quantification of collagen as described chimera (R&D systems, Minneapolis, MN) was then added in binding previously (Koobatian et al., 2016). Briefly, tissues used for quantification buffer for 1 h at 37°C (Lira et al., 2008). CDH11-Fc consisted of the five of elastin and collagen were lyophilized to obtain the dry weight, which extracellular domains of CDH11 linked to the constant heavy chain domains served as a normalization factor. These were then boiled at 95°C for 45 min CH2 and CH3 of FCγ through the hinge region, which existed in solution as in 0.1 M NaOH to yield cross-linked elastin as an insoluble residue. The a homodimer with disulfide bridges between the monomeric subunits (see pellet and supernatant were separated by centrifugation and the supernatant Fig. 4A). A similar method was followed for CDH2-Fc-coated plates (R&D was reserved for collagen quantification. The pellet was then dried again for systems, Minneapolis, MN). When cells were plated on this surface, acid hydrolysis, using 6 M HCl, overnight at 105°C. Lyophilized homophillic interactions occurred between cell surface cadherins and hydrolyzed protein was resuspended in water and samples at the desired exposed homodimers of CDH11-Fc. An optimized cell density of 7000 dilution and standards (Alpha elastin, EPC) were loaded into 96-well plates. cells/cm2 was used in order to avoid cell–cell interactions and single out the Ninhydrin reagent (Sigma) was then added and the plates were incubated for effect of CDH11 contacts between the cells and the CDH11-Fc surface. 40 min at 65°C. Plates were then read at 550 nm using a plate reader (Synergy 4, BioTek, Winooski, VT). Fibrin gel compaction The reserved supernantant was also subjected to acid hydrolysis after Fibrin gels were formed around a mandrel as previously described (Swartz lyophilization. The protocol for the hydroxyl-proline assay is as described et al., 2005; Liu et al., 2007a). Following polymerization at 37°C for 1 h, elsewhere (Liang et al., 2013). gels were released from the wall and images of wells were obtained at indicated times. Experimental triplicates or quadruplicates with each group Histological examination of tissues were used to measure area of fibrin gel as a fraction of total area (A/A0)ofa 2 Pressure-fixed samples were dehydrated in a series of graded ethanol standard 24-well plate (A0=2 cm ). These gels were further cultured in solutions and xylene substitutes and then embedded in paraffin as reported vessel medium (Liang et al., 2013) in the same plates for 14 days, to allow before (Geer et al., 2002; Swartz et al., 2005). For histological evaluation, 5- for collagen deposition and remodeling. μm paraffin-embedded tissue sections were stained with Masson’s trichrome or Verhoeff’s elastin using a Masson trichrome staining or Elastic stain qRT-PCR kit (Chromaview, Richard-Allen scientific, Kalamazoo, MI), following the Total mRNA was extracted from cell monolayers by using an RNeasy Mini manufacturer’s directions. Kit (Qiagen, Balencia, CA). Single-strand cDNA was reverse transcribed and synthesized from purified 1.0 µg mRNA using the QuantiTect Reverse Cell isolation and culture Transcription Kit (Qiagen, Balencia, CA). Real-time PCR analysis was Human neonatal foreskin fibroblasts and mice dermal fibroblasts were performed in CFX96 real-time system (Bio-Rad, Hercules, CA) with a isolated from tissues as reported elsewhere (Bajaj et al., 2001). Mice aortic mixture of iQ SYBR Green Supermix (Bio-Rad, Hercules, CA), primers ’ smooth muscle cells (ASMCs) were isolated from mice aorta sections (Table S3) and cDNA prepared by following manufacturer s instructions. Δ as described previously (Swartz et al., 2005). Human dermal fibroblasts were Gene expression levels were quantified and analyzed using the CT method, cultured in Dulbecco’smodifiedEagle’s medium (DMEM) supplemented and are reported as normalized to the housekeeping gene (RPL32 for human Gapdh with antibiotic-antimycotic cocktail and 10% MSC-qualified fetal samples and for mice). bovine serum, supplemented with 2 ng/ml basic fibroblast growth factor (bFGF, all components from Life Technologies, Burlington, ON, Canada). Western blotting Passages between 2 and 12 were used in all experiments. Mice dermal Protein lysates were isolated from pre-incubated cell monolayers in standard fibroblasts and ASMCs were cultured in DMEM supplemented with 10% lysis buffer at the indicated times. The lysates were subjected to western blot MSC-qualified fetal bovine serum, penicillin-streptomycin-glutamine and β- analysis as described previously (Alimperti et al., 2014). Primary antibodies mercaptoethanol (Life Technologies), non-essential amino acids and essential (Table S4) were incubated overnight at 4°C followed by washes and Journal of Cell Science

2959 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 2950-2961 doi:10.1242/jcs.183772 incubation with secondary antibody at room temperature for 1 h. Visualized Cavallaro, U. and Dejana, E. (2011). Adhesion molecule signalling: not always a bands were developed with LumiGLO reagent (Cell Signaling sticky business. Nat. Rev. Mol. Cell Biol. 12, 189-197. Technologies, Beverly, MA) according to the manufacturer’s protocol. Diaz-Chavez, J., Hernandez-Pando, R., Lambert, P. F. and Gariglio, P. (2008). Down-regulation of transforming growth factor-beta type II receptor (TGF-betaRII) Protein content was analyzed by densitometric analysis using Image J and protein and mRNA expression in cervical cancer. Mol. Cancer 7,3. results are reported as normalized to GAPDH. Geer, D. J., Swartz, D. D. and Andreadis, S. T. (2002). Fibrin promotes migration in a three-dimensional in vitro model of wound regeneration. Tissue Eng. 8, 787-798. Immunohistochemistry Goepel, C. and Thomssen, C. (2006). Changes in the extracellular matrix in Cell monolayers, at indicated times, were fixed with 4% paraformaldehyde for periurethral tissue of women with stress urinary incontinence. Acta Histochem. 15 min at room temperature. Following permeabilization with 0.1% Triton X- 108, 441-445. Harris, T. J. C. and Tepass, U. (2010). Adherens junctions: from molecules to 100 in PBS and incubation in blocking buffer (5% goat serum in PBS) at room morphogenesis. Nat. Rev. Mol. Cell Biol. 11, 502-514. temperature for 1 h, primary antibodies were added in dilutions as mentioned Itoh, Y., Kimoto, K., Imaizumi, M. and Nakatsuka, K. (2007). Inhibition of RhoA/ (Table S5). After incubation with primary antibodies overnight at 4°C, Rho-kinase pathway suppresses the expression of type I collagen induced by secondary antibody (1:200 in PBS, Alexa-Fluor-488-conjugated goat anti- TGF-beta2 in human retinal pigment epithelial cells. Exp. Eye Res. 84, 464-472. rabbit IgG, Life Technologies, Burlington, ON, Canada) was added for 1 h at Johnson, L. A., Rodansky, E. S., Haak, A. J., Larsen, S. D., Neubig, R. R. and – Higgins, P. D. R. (2014). Novel Rho/MRTF/SRF inhibitors block matrix-stiffness room temperature. Phalloidin Alexa-Fluor-594 (Life Technologies) was used – ’ and TGF-beta induced fibrogenesis in human colonic myofibroblasts. Inflamm. to stain for F-actin as per the manufacturer s instructions. Cell nuclei were Bowel Dis. 20, 154-165. counterstained with Hoechst 33342 (10 mg/ml; 1:200 dilution; 5 min at room Kawaguchi, J., Azuma, Y., Hoshi, K., Kii, I., Takeshita, S., Ohta, T., Ozawa, H., temperature; EMD Millipore Laboratory Chemicals, Billerica, MA). Takeichi, M., Chisaka, O. and Kudo, A. (2001a). Targeted disruption of Fluorescence microscopy images were acquired using the Zeiss Axiovision cadherin-11 leads to a reduction in bone density in calvaria and long bone observer Z1 (LSM 510; Zeiss, Oberkochen, Germany) equipped with a digital metaphyses. J. Bone Miner. Res. 16, 1265-1271. camera (ORCA-ER C4742-80; Hamamatsu, Bridgewater, NJ) and images Kawaguchi, J., Kii, I., Sugiyama, Y., Takeshita, S. and Kudo, A. (2001b). The transition of cadherin expression in osteoblast differentiation from mesenchymal were analyzed using the ImageJ software. cells: consistent expression of cadherin-11 in osteoblast lineage. J. Bone Miner. Res. 16, 260-269. Statistical analysis Kimura, Y., Matsunami, H., Inoue, T., Shimamura, K., Uchida, N., Ueno, T., Values are mean±s.d. Significant differences between animal groups were Miyazaki, T. and Takeichi, M. (1995). Cadherin-11 expressed in association with determined by Student’s t-test (unpaired, two-tailed) or ANOVA with post- mesenchymal morphogenesis in the head, , and limb bud of early mouse hoc analysis using Student–Newman–Keuls multiple comparison test or . Dev. Biol. 169, 347-358. Kitchen, C. M., Cowan, S. L., Long, X. and Miano, J. M. (2013). Expression and Spearman correlations analysis. promoter analysis of a highly restricted alpha gene in vascular smooth muscle. Gene 513, 82-89. Competing interests Kohl, E., Steinbauer, J., Landthaler, M. and Szeimies, R.-M. (2011). Skin ageing. The authors declare no competing or financial interests. J. Eur. Acad. Dermatol. Venereol. 25, 873-884. Koobatian, M. T., Row, S., Smith, R. J., Jr., Koenigsknecht, C., Andreadis, S. T. Author contributions and Swartz, D. D. (2016). Successful endothelialization and remodeling of a cell- S.R. and Y.L. contributed equally as first authors. They participated in primary data free small-diameter arterial graft in a large animal model. Biomaterials 76, collection, statistical analyses, intellectual contribution and preparation and editing 344-358. of the manuscript. S.A. contributed intellectually and in primary data collection for Leitner, L., Shaposhnikov, D., Mengel, A., Descot, A., Julien, S., Hoffmann, R. mice tissues as well as preparation of plasmids. S.K.A. maintained and bred the and Posern, G. (2011). MAL/MRTF-A controls migration of non-invasive mice at Baylor College of Medicine, contributed towards mice tissue and data cells by upregulation of -associated proteins. J. Cell Sci. 124, collection and cell isolation and participated in editing the manuscript. S.T.A. is the 4318-4331. corresponding author and contributed intellectually to the entire study and Li, G.-Y., Cui, W.-S., Zhou, F., Gao, Z.-Z., Xin, H., Liu, T., Li, W.-R., Gong, Y.-Q., specifically to experimental design, data interpretation and writing of the manuscript. Bai, G.-Y., Guo, Y.-L. et al. (2012a). Pathology of urethral fibromuscular system He is also responsible for management of resources and planning of the study. related to parturition-induced stress urinary incontinence and TGF-beta1/Smad S.K.A. and S.T.A. contributed to grant acquisition and management. All authors pathway. Mol. Cell. Biochem. 364, 329-335. agree to the authorship as listed in the manuscript. Li, L., Bennett, S. A. and Wang, L. (2012b). Role of E-cadherin and other cell adhesion molecules in survival and differentiation of human pluripotent stem cells. Cell. Adh. Migr. 6, 222-233. Funding Liang, M.-S., Koobatian, M., Lei, P., Swartz, D. D. and Andreadis, S. T. (2013). This study was supported by the National Institutes of Health (NIH) [grant number Differential and synergistic effects of mechanical stimulation and growth factor R01 AR062056-05 to S.K.A.]; and the National Science Foundation (NSF) [grant presentation on vascular wall function. Biomaterials 34, 7281-7291. number CBET 1403086 to S.T.A.]. Deposited in PMC for release after 12 months. Lira, C. B. B., Chu, K., Lee, Y.-C., Hu, M. C.-T. and Lin, S.-H. (2008). Expression of the extracellular domain of OB-cadherin as an Fc fusion protein using bicistronic Supplementary information retroviral expression vector. Protein Expr. Purif. 61, 220-226. Supplementary information available online at Liu, J. Y., Swartz, D. D., Peng, H. F., Gugino, S. F., Russell, J. A. and Andreadis, http://jcs.biologists.org/lookup/doi/10.1242/jcs.183772.supplemental S. T. (2007a). Functional tissue-engineered blood vessels from bone marrow progenitor cells. Cardiovasc. Res. 75, 618-628. References Liu, W. 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