Semaphorin 4A enhances lung fibrosis through activation of Akt via PlexinD1 receptor

1,† 1,† 2 3 4, HAI-YING PENG , WEI GAO , FA-RONG CHONG , HONG-YAN LIU and JI ZHANG * 1Department of Clinical Laboratory Medicine, 2Department of Pediatric Surgery, and 3Department of Endocrinology, Linyi People’s Hospital, Linyi, Shandong 276003, China 4Department of Thoracic Surgery and Lung Transplantation Center, Wuxi People’s Hospital Affiliated to Nanjing Medical University, Wuxi, Jiangsu 214123, China

*Corresponding author (Email, [email protected]) †These authors contributed equally to the work.

Semaphorin 4A plays a regulatory role in immune function and angiogenesis. However, its specific involvement in controlling lung fibrosis, a process that is closely related to angiogenesis and inflammation is still poorly understood. In the present study, we show that treatment of Sema4A on normal lung fibroblasts induces expression of proteins that contribute to a contractile phenotype, including α-smooth muscle actin (α-SMA), ezrin, moesin, and paxillin. We confirm that Sema4A enhances the ability of lung fibroblasts to contract collagen gel. Sema4A treatment led to resistance to apoptosis in normal lung fibroblasts. Relative to normal lung fibroblasts, fibroblasts cultured from scars of patients with the fibrotic disease Systemic Sclerosis (SSc) showed elevated Sema4A secretion, enhanced α-SMA, ezrin, moesin, and paxillin expression, and high ability to induce collagen gel contraction. Using neutralizing antibody against Sema4A receptor, PlexinD1, we found that endogenous Sema4A signalling in SSc fibroblast was through PlexinD1 receptor. We then identified the signalling mechanism through which Sema4A-PlexinD1 promotes the ability of normal fibroblasts to contract a collagen gel matrix. Western blot analysis showed that Sema4A activated the Akt pathway in lung fibroblasts, and the specific inhibitor of Akt pathway, Akt inhibitor III, blocked the ability of Sema4A to promote the ability of lung fibroblasts to contract a collagen gel matrix. Thus, blocking Sema4A- PlexinD1-Akt cascades might be beneficial in reducing pulmonary fibrosis.

[Peng H-Y, Gao W, Chong F-R, Liu H-Y and Zhang J 2015 Semaphorin 4A enhances lung fibrosis through activation of Akt via PlexinD1 receptor. J. Biosci. 40 855–862] DOI 10.1007/s12038-015-9566-9

1. Introduction Suzuki et al. 2008). A membrane-bound class 4 Semaphorin, Sema4A, has been reported to progressively increase its expres- Semaphorins compose a large family of secreted and sion in developing mouse embryos (Püschel et al. 1995) and membrane-bound glycoproteins that are divided into eight become prominent in the adult brain, lung, kidney, spleen, testis, subclasses, 1 to 7 and viral (Kumanogoh and Kikutani 2003; and mammary gland (Kumanogoh et al. 2002). Sema4A has Suzuki et al. 2008). They were first identified in the nervous been proven to play a regulatory role in immune function, such system with a defined function as axon guidance molecules. as Th1/Th2 differentiation and inflammation (Kumanogoh et al. All semaphorins contain a homologues sequence of appro- 2005). PlexinD1 has been identified as one of receptors of ximately 500 aa in the N-terminal extracellular domain. Sema4A, and upon binding to PlexinD1, Sema4A exerts a The most characterized receptors for semaphorins are proangiogenic effect by enhancing vascular endothelial growth plexins and neuropilins (Kumanogoh and Kikutani 2003; factor-A expression in macrophages (Meda et al. 2012). The

Keywords. Akt signalling; fibrosis; lung fibroblast; PlexinD1; Semaphorin 4A http://www.ias.ac.in/jbiosci J. Biosci. 40(5), December 2015, 855–862, * Indian Academy of Sciences 855

Published online: 28 November 2015 856 Hai-Ying Peng et al. evidences highlight the important role played by Sema4A in Aldrich, St Louis, MO) on ice, neutralizing with 1 M NaOH regulating the immune system function and angiogenesis. How- and diluting to 1 mg/mL collagen. Fibroblasts were ever, its specific involvement in controlling lung fibrosis, a trypsinized, resuspended in cold, serum-free DMEM, and process that is closely related to angiogenesis and inflammation, added to the collagen mixture at a final concentration of is still poorly understood. 5×105 cells/mL with 0.75 mg/mL as final collagen concen- Lung fibrosis, a pathological process resulting in tissue re- tration (Navarro et al. 2009). Aliquots (in triplicate) were modelling from overabundant extracellular matrix deposition, is cast onto 24-well plates and allowed to solidify at 37°C (30– a major cause of morbidity and mortality. Myofibroblasts are 45 min), after which serum-free media was added, incubated. considered as the chief perpetrators of this pathology. The area of the collagen gels was measured by Leica Myofibroblasts are a trans-differentiated fibroblast phenotype, image analyser system (LEICA Microsystems, Germany). identified by alpha smooth muscle actin (α-SMA) expression The images were observed and recorded. The area of the (Hinz et al. 2001; Hinz and Gabbiani 2003). They are avid gels was processed by computer software from the extracellular matrix synthesizers and have enhanced collagen microsystems, and gel contraction in the horizontal axes contraction ability. Myofibroblasts are more resistant to apopto- was determined. The gel area of control group in each tic signalling/induction than fibroblasts (Nyp et al. 2014), which experiment was set as 100%. is particularly important because a failure of proper apoptotic termination – a consequence of an inability of epithelial cells to successfully restore damaged epithelium – is a significant con- 2.3 Western blot analysis tributor to pathological fibrosis. Regulation of fibroblast acqui- sition of this trans-differentiated phenotype is a complex process To examine biochemical or functional differences between modulated at multiple levels: transcription, translation, and post- control and Sema4A treated lung fibroblasts, a series of translation (Bell et al. 1979; Gadek et al. 1984; Tomasek et al. Western blot experiments were performed. Fibroblasts 2002). The persistence of the myofibroblasts within the fibrotic were cultured and treated with Sema4A in collagen gels lesion results in elevated levels of matrix synthesis and contrac- as described above. Alternatively, fibroblasts were grown tion, causing scar formation (Schmitt-Graff et al. 1994) and to confluence in DMEM with 10% fetal calf serum and functional impairment of the affected organ (Panos et al. 1990). then serum starved in DMEM with 0.5% bovine serum In this study, we investigated the role of Sema4A- albumin (BSA) for 24 h. After serum starvation, cells were PlexinD1-Akt pathway in regulating fibroblast/ stimulated with 900 ng/mL Sema 4A for 24 h with 0.5% myofibroblast trans-differentiation during lung fibrosis. Our BSA. Cell layer lysates were examined. SDS-PAGE was findings may have implications for the development of po- performed on 12% polyacrylamide gels, and the separated tential therapies for pathological fibrosis. proteins were transferred onto nitrocellulose membranes at 30 V for 90 min. Membranes were blocked by incubation for 1 h with 5% nonfat milk in TBS containing 0.2% 2. Materials and methods Tween 20, and antigens were detected using specific anti- bodies. Cell layer lysates (10 μg/sample) were probed 2.1 Patients, cells and reagents using antibodies directed against α-SMA (Sigma-Aldrich), Akt, p-Akt, anti-paxillin, anti-ezrin, anti-moesin, Anti- Fibroblasts were grown from lung biopsy specimens from Caspase 3, anti-Caspase 8, anti-Akt, anti-phosphoAkt, an- SSc patients. This study was approved by local ethics com- ti-ERK, anti-phospho-ERK, anti-p38 and anti-p hospho mittee of Linyi People’s hospital and carried out in accor- p38 (Cell Signaling Technology), anti-PlexinD1 (Abcam), dance with the approved guidelines. Human Lung and anti-GAPDH (Sigma), followed by incubation with Fibroblasts Adult (HLF-A) origin were purchased from Cell appropriate horseradish peroxidase-conjugated bound sec- Applications, Inc. (San Diego, CA) and grown in HLF ondary antibody (Jackson Immuno Research Laboratories). growth media (used passages 3–5). Recombinant human Signal was detected using the enhanced chemilumines- Sema4A was from ACRO Biosystems (Newark, Delaware). cence protocol (Pierce) according to manufacturer’s Akt inhibitor IIIwas from Santa Cruz Biotechnology. instructions. Densitometry of Western blot was analysed PlexinD1 neutralizing antibody was purchased from R&D by using Image J software. Systems. 2.4 Flow cytometry 2.2 Collagen contraction Apoptosis of normal lung fibroblasts was measured with Type I collagen gels were prepared by mixing cold rat tail PE-annexin-V apoptosis detection kit (BD Biosciences) HC collagen (BD Biosciences) with 10X DMEM (Sigma- according to the manufacturer. Briefly, cell were washed

J. Biosci. 40(5), December 2015 Semaphorin 4A enhances lung fibrosis 857 with PBS, resuspended in provided 1X binding buffer and 3. Results incubated with Annexin V-PE and 7-AAD for 15 min prior to analysis. Cells were then immediately analyzed by flow 3.1 Sema4A induces expression of procontractile cytometer (BD LSRII). proteins and enhances collagen contraction in lung fibroblasts 2.5 ELISA of Sema4A in systemic sclerosis (SSc) lung fibroblast supernatant To investigate the role of Sema4A in fibroblast biology, we starved normal human lung fibroblasts overnight and Sema4A secretion was measured in supernatants collected from then treated cells with or without 900 ng/mL Sema4A. We cultures of normal and SSc lung fibroblasts in serum-free examined whether Sema4A treatment could alter expression medium, by using Sema4AELISA kit according to manufac- of α-SMA, ezrin, moesin proteins. Furthermore, paxillin, turer’s instructions (Cloudy Clone, Corp. Texas, USA). The a protein found in focal adhesions, whose recruitment values were given as Sema4A per milliliter per million cells. to the cellular membrane is critical for the development of tension during smooth muscle contraction (Navarro 2.6 Statistical analysis et al. 2009), was examined. We found that Sema4A treat- ment induced α-SMA, ezrin, moesin, and paxillin expression Results are expressed as means±SD of data obtained. Statis- in lung fibroblasts (figure 1A). Also, Sema4A treatment tical analysis was performed with Student’s t-test. A value of dramatically enhanced collagen contraction ability of lung P<0.05 or P<0.01 was considered significant. Results are fibroblast (figure 1B). Thus, we concluded that Sema4A representative of 3 independent experiments. might contribute to the contractile phenotype of fibroblasts.

Figure 1. Sema4A induces α-SMA, ezrin, moesin, and paxillin expression and collagen gel contraction in normal human lung fibroblasts. (A) Western blots of α-SMA, ezrin, moesin, and paxillin expression in normal human lung fibroblasts in the presence of 900 ng/mL Sema4A. (B) Induction of collagen gel contraction by Sema4A in normal lung fibroblasts. *P<0.05 and **P<0.01 and results are mean ± S.D. Results are representative of 3 independent experiments.

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Figure 2. Sema4A treatment leads to decreased apoptosis of human lung fibroblasts. (A) Protein levels of cleaved Caspase 3 and 8 were examined by Western blot in normal human lung fibroblasts in the presence of 900 ng/mL Sema4A. Normal cells with no Sema4A treatment were set as control. (B) Analysis of apoptosis by Flow cytometry in human lung fibroblasts treated with 900 ng/mL Sema4A and control cells.*P<0.05 and **P<0.01. Results are mean ± S.D. Results are representative of 3 independent experiments.

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3.4 Sema4A promotes collagen contraction in human lung fibroblasts through PlexinD1 receptor

We then investigated whether lung fibroblasts cultured from patients of SSc fibrotic lung disease displayed an enhanced contractile phenotype relative to normal lung fibroblasts. We found that compared to the normal lung fibroblasts, SSc lung fibroblast expressed higher levels of α-SMA, ezrin, moesin, and paxillin (figure 4A). We further sought to determine whether this elevated contractile phenotyoe was caused by elevated level of Sema4A expressed by SSc fibroblasts. To perform this analysis, we compared the ability of SSc and normal lung fibroblasts to contract a collagen gel matrix over Figure 3. Sema4A levels in the supernatants from normal and SSc a 24 h period. As shown in figure 4B, SSc lung fibroblasts lung fibroblasts. Medium conditioned for 24 h by normal and SSc showed a significantly increased ability to contract a colla- lung fibroblasts was examined. The concentration of Sema4A in gen matrix, relative to normal lung fibroblasts. To assess conditioned media was measured by an ELISA that detects Sema4A levels. **P<0.01 and results are mean ± S.D. Results are represen- whether this ability of SSc lung fibroblasts could be due to tative of 3 independent experiments. elevated, constitutive Sema4A signalling, we tested the ef- fect of neutralizing antibody against one of Sema4A recep- tors, PlexinD1, on the contractile phenotype of SSc lung fibroblasts. We found that PlexinD1-neutralizing antibody potently reduced the contractile ability of SSc lung fibro- blasts (figure 4B).We also observed that PlexinD1- 3.2 Sema4A treatment leads to resistance to apoptosis neutralizing antibody significantly blocked Sema4A en- in human lung fibroblasts hanced collagen contraction ability of normal lung fibroblast (figure 4C). The ability of myofibroblasts to resist apoptosis signalling is linked to the development of pathological fibrosis, while the termination of fibroblast contractility response is im- 3.5 Activated Akt signalling pathway mediates Sema4A- portant for nonfibrotic wound healing (Nyp et al. 2014). PlexinD1-enhanced collagen contraction in human lung To assess whether Sema4A could affect fibroblast apopto- fibroblasts sis response, cleaved Caspase 3 and Caspase 8 were de- tected by Western blot in samples from cells embedded in We then sought to identify the signalling pathway through collagen gels (figure 2A). As shown in figure 2A, expres- which Sema4A-PlexinD1 promotes the ability of normal sion of cleaved Caspase 3 and Caspase 8 was reduced in fibroblasts to contract a collagen gel matrix. Western blot the cells treated with Sema4 compared with control cells. analysis with anti-phospho-ERK, anti-phospho-p38, and We examined the percentage of apoptotic lung fibroblasts anti-phospho-Akt antibodies showed that Sema4A activated in the presence of Sema4A and found that compared to Akt pathway in lung fibroblasts (figure 5A). We then found negative control cells, Sema4A remarkably reduced per- specific inhibitor of Akt pathway, Akt inhibitor III, blocked centage of apoptotic cells and leads to resistance to apo- the ability of Sema4A to promote the ability of lung fibro- ptosis (figure 2B). blasts to contract a collagen gel matrix (figure 5B).

3.3 SSc lung fibroblasts show elevated levels 4. Discussion of Sema4A expression In the present investigation, human lung fibroblasts were To assess whether SSc lung fibroblasts secrete elevated utilized as an in vitro model to examine the role of Sema4A levels of Sema4A, we examined Sema4A levels in superna- in ECM remodelling and fibrosis. It is generally considered tants from normal and SSc lung fibroblasts. We plated equal that the excessive fibrogenesis and the resultant extracellular numbers of SSc and normal fibroblasts, and after 24 h, equal matrix remodelling in pathological fibrosis are due to the amounts of media were used for measuring secreted Sema4A presence and activity of activated fibroblasts of the levels. We found that SSc lung fibroblasts secreted 3 times myofibroblast phenotype and function (Kis et al. 2011). the amount of Sema4A relative to normal lung fibroblasts Fibroblasts of the myofibroblast phenotype are generally (figure 3). considered to be more resistant to apoptosis and have

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Figure 4. SSc lung fibroblasts express higher levels of α-SMA, ezrin, moesin, and paxillin , and exhibit stronger ability to contract collagen gel than that of normal lung fibroblasts. PlexinD1-neutralizing antibody blocks the ability of SSc lung fibroblasts to contract collagen gel. (A) Western blots of α-SMA, ezrin, moesin, and paxillin expression in normal human lung fibroblasts and SSc lung fibroblasts. (B) Comparison of collagen gel contraction by normal lung fibroblasts, SSc fibroblasts and SSc fibroblasts treated with 5 μg/mL neutralizing antibody against Sema4A receptor, PlexinD1. (C) Comparison of collagen gel contraction by normal lung fibroblasts treated with 900 ng/mL Sema4A for 1h in the presence of 5 μg/mL neutralizing antibody against Sema4A receptor, PlexinD1. *P<0.05 and **P<0.01. Results are mean ± S.D. Results are representative of 3 independent experiments.

increased collagen contraction ability. The ability of levels in SSc lung fibroblasts and Sema4A level in superna- myofibroblasts to resist apoptosis has been suggested as a tant of SSc lung fibroblast. We found that, relative to normal central event driving the development of fibrotic disease, lung fibroblasts, SSc fibroblasts produced elevated levels of while appropriate termination of activated fibroblast- α-SMA, ezrin, moesin, and paxillin, and secreted higher mediated response leads to normal fibrogenesis. In the cur- levels of Sema4A. We then found that endogenous Sema4A rent work, we report that Sema4A treatment on human lung in lung SSc fibroblasts directly contributed to the contractile fibroblasts has the promotive effect of inducing trans- phenotype of the cells as evidenced by the fact that blockade differentiation to the myofibroblast phenotype and function of Sema4A signalling by neutralizing antibody against by inducing α-SMA expression, resistance to apoptosis and Sema4A receptor, PlexinD1 dramatically reduce the ability an increased ability for collagen contraction. of SSC fibroblasts to contract collagen gel matrix led by To assess whether Sema4A could be involved in en- Our findings proved that Sema4A-PlexinD1 signalling can hanced collagen contraction by lesional SSc pulmonary fi- induce trans-differentiation of lung fibroblast to myofibroblast. broblasts, we examined α-SMA, ezrin, moesin, and paxillin It has been shown that Akt regulates myocardial contraction

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Figure 5. Sema4A induces Akt phosphorylation, but not phosphorylation of ERK and p38 in normal lung fibroblasts. Akt inhibitor III reduced the collagen gel contraction in SSc lung fibroblasts. (A) Normal lung fibroblasts were cultured, serum starved for 18 h, and treated with 900 ng/mL Sema4A. Western blot analysis was performed with anti-ERK, anti-phospho-ERK, anti-p38, anti-phospho-p38, anti-Akt and anti-phospho-Akt antibodies. (B) Normal lung fibroblasts were pretreated with 1 μM Akt inhibitor III for 2 h, and then treated with 900 ng/mL Sema4A for 1 h. Comparison of collagen gel contraction by normal lung fibroblasts in the presence of Sema4A or the combination of Sema4A and Akt inhibitor III was performed. *P<0.05 and results are mean ± S.D. Results are representative of 3 independent experiments.

(Condorelli et al. 2002) and is activated during contraction of confirm whether Sema4A induced elevated collagen gel skeletal and smooth muscle (Sakamoto et al. 2003). Thus, contraction in normal lung fibroblast is Akt-pathway- activation of the Akt pathway may be generally required for dependent. We confirmed that Akt inhibitor III significantly actin-generated contractile forces. It has also been reported that reduced the ability to induce collagen gel contraction of in addition to activation of Akt pathway, ERK and P38 normal lung fibroblast treated with Sema4A. Thus, it is pathways are also beneficial in scar formation in pulmonary suggested that Sema4A-PlexinD1-Akt signalling pathway fibrosis (Shi-Wen et al. 2004). In our study, we showed that may play an important role in lung fibrosis. only phospho-Akt was elevated in response to Sema4D treat- Our findings revealed that Sema4A plays a regulatory role ment in normal lung fibroblast. Akt inhibitor III was utilized to in modulating fibroblast acquisition of the phenotype and

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MS received 08 April 2015; accepted 05 August 2015

Corresponding editor: SORAB NDALAL

J. Biosci. 40(5), December 2015