Regulation of Fibroblast Cell Polarity by Src Tyrosine Kinase

Src protein tyrosine kinases (SFKs) are a family of nonreceptor tyrosine kinases that are localized beneath the plasma membrane and are activated during cell adhesion, migration, and elongation. Due to their involvement in the activation of signal transduction cascades, SFKs have been suggested to play important roles in the determination of cell polarity during cell extension and elongation. However, the mechanism underlying Src-mediated polarity formation remains unclear. The present study was performed to investigate the mechanisms underlying Src-induced cell polarity formation and cell elongation using Src knockout fibroblasts (SYFs) together with an inhibitor of Src. Normal and Src knockout fibroblasts were also transfected with a wild-type c-Src, dominant negative c-Src, or constitutively active c-Src gene to analyze the changes in cell morphology. SYF cells cultured on a glass substrate elongated symmetrically into spindle-shaped cells, with the formation of focal adhesions at both ends of the cells. When normal fibroblasts were treated with Src Inhibitor No. 5, a selective inhibitor of Src tyrosine kinases, they elongated into symmetrical spindle-shaped cells, similar to SYF cells. These results suggest that cell polarity during extension and elongation may be regulated by SFKs and that the expression and regulation of Src are important for the formation of polarity during cell elongation.


Introduction
Cell polarity refers to the spatial, morphological, and structural asymmetry of cells and involves a characteristic spatial distribution of morphological, structural, and cellular components, i.e., the plasma membrane, cytoskeletal components, and sites of cell-cell adhesion (focal adhesions). Cell functions, such as transport, intracellular signaling, and mechanosensing, are dependent on the asymmetrical distribution of intracellular components. For example, epithelial cell polarity has an apical membrane-outer basal membrane orientation, and neural cell polarity is involved in the formation of two types of neurites, i.e., dendrites and axons [1]. Adhesive structures, adherens junctions, tight junctions, and focal adhesions allow the formation of two distinct compartments, i.e., the apical membrane that normally faces the external environment and the basolateral membrane that is connected to the extracellular matrix (ECM) of the basement membrane via integrins and their receptors.
Actin filaments are major components of actomyosin contractile systems in eukaryotic cells that regulate the direction of cell movement. Actin molecules switch between polymerization and depolymerization with the activation of Rho GTPases, a family of small G proteins, and their downstream molecules (the WASP/WAVE family and the Arp2/3 complex) [2,3]. Thus, stress fibers, lamellipodia, and pseudopodia, which control cell morphology and plasma membrane dynamics via actomyosin contractile systems, function in determining the polarization of cells [4].
When cells are cultured on a glass substrate, the plasma membrane begins to move in from the distal end to the leading edge of the cell [5]. Depolymerization of the actin cytoskeleton deforms the morphology of the cell membrane, and the focal adhesions between the extracellular matrix and intracellular proteins move forward to the leading activity of the Src family at focal adhesions regulates the metabolic turnover of focal adhesions during cell motility, resulting in the formation of cell polarity. As a result, Src exerts diverse physiological functions, such as activation of cell adhesion and motility, differentiation, proliferation, survival, and even induction of transformation [29,33]. The present study was performed to examine the effects of the expression of activated or dominant negative forms of the c-Src gene in fibroblasts to gain insight into the role of Src tyrosine kinase in cell motility, especially in the establishment of cell polarity.
Fibroblasts were treated with Src Inhibitor No. 5, a selective inhibitor of Src tyrosine kinases, to block Src activation and allow us to investigate its influence on the physiological characteristics of the cells and elucidate the mechanisms underlying polarized elongation. Normal fibroblasts and Src family knockout cells [34] (SYF cells; ATCC, Manassas, VA) were also transfected with constructs encoding wild-type c-Src (WT c-Src), constitutively active c-Src, and dominant negative c-Src genes to analyze the changes in cell morphology. In this study, SYF cells transfected with WT c-Src showed an elongated morphology while extending pseudopodia similar to normal fibroblasts. Normal fibroblasts transfected with the dominant negative c-Src mutant gene showed symmetrical spindle-shaped morphology. Normal fibroblasts treated with the c-Src-specific inhibitor, Src Inhibitor No. 5 (Biaffin, Kassel, Germany), adopted a symmetrical spindle shape similar to SYF cells. These observations suggest that SFKs regulate polarity formation during cell extension and elongation and that the expression and regulation of at least one member of the SFKs, c-Src, are important for polarity formation during cell elongation.

Immunofluorescence Microscopy
Cultured cells were fixed with 1% paraformaldehyde in PBS for 30 min and permeabilized by treatment with 0.05% Triton X-100 in PBS for 5 min. The fixed cells were incubated with 10% normal goat serum for 30 min at room temperature and then stained with an antibody against vinculin (Sigma, St. Louis, MO, USA) as a marker of focal adhesions for 60 min. After washing in PBS for 20 min, the fixed specimens were incubated with fluorescein-conjugated anti-mouse IgG. Samples were then observed by conventional epifluorescence microscopy or phase-contrast microscopy (Olympus, Tokyo, Japan).

SFK Inhibitor
The SFK inhibitor, Src Inhibitor No. 5 (Biaffin), is one of several Src inhibitors that belong to the quinazoline class of selective inhibitors of c-Src and has an IC 50 of 10 nM in vitro [35][36][37]. Fibroblasts were seeded onto dishes and cultured for 24 h, followed by incubation with Src Inhibitor No. 5 at a concentration of 10 µM. Dominant negative c-Src (K295M/Y527F) and constitutively active c-Src (Y527F; chicken c-Src residue numbering) were generated by site-directed mutagenesis using a KOD-plus mutagenesis kit, in accordance with the manufacturer's protocol (Toyobo, Tokyo, Japan), and cloned into a cytomegalovirus (CMV) promoter-driven pUSEamp-WT Src expression vector (Upstate Biotechnology, Lake Placid, NY, USA). WT Src or dominant negative c-Src (K295M/Y527F) was inserted into peGFP-C1 (Clontech, Palo Alto, CA, USA). eGFP-fused dominant negative c-Src (K295M/Y527F) and WT c-Src were transfected into fibroblasts using Tfx-50, in accordance with the manufacturer's protocol (Promega, Madison, WI, USA). pUSEamp-dominant negative c-Src (K295M/Y527F), constitutively active c-Src (Y527F), or WT c-Src was also transfected into fibroblasts using Tfx-50 (Promega), in accordance with the manufacturer's protocol. Cells transfected with eGFP-C1 or pUSEamp(−) alone did not show any differences in morphology from normal fibroblasts. Neomycinresistant cells were selected by growth in 400 µg/mL G-418 and maintained in 200 µg/mL G-418. G-418 was removed prior to the experiments. Control cells were transfected with vector alone, cloned, and treated in a manner similar to that described for the experimental cells. Transfected cells were plated on glass-bottomed culture dishes and placed on a temperature-controlled stage at 37 • C (Matsunami, Tokyo, Japan). The cell-substrate interface was examined by conventional phase-contrast microscopy and epifluorescence microscopy (Olympus).

Calculation of the Vertical Axis of the Cell and the Aspect Ratio
The vertical axis and aspect ratio of 50 cells were measured using open-source Fiji image analysis software [38]. Statistical analyses were performed using Excel (Microsoft, Redmond, WA, USA). Unpaired two-tailed Student's t-test was used to compare the means between the two groups. All statistical tests were two-sided, and p < 0.05 was taken to indicate statistical significance.

Results
SFKs are nonreceptor tyrosine kinases that play key roles in the regulation of signal transduction. SFK activation and protein levels are elevated in various types of cancer, and there has been a great deal of research regarding the regulation of Src kinase activity. SFKs consist of several proteins, i.e., Src, Fyn, Yes, Fgr, Lck, Hck, Blk, Lyn, Frk, and Yrk, that interact with the intracellular domains of growth factors/cytokine receptors, G proteincoupled receptors (GPCRs), and integrins [39][40][41][42]. Members of the SFK family have similar domain structures, and several small molecule inhibitors that show selectivity for SFKs are available.
When cultured on a glass substrate, SYF cells first adopted an extended pancake shape and then spread out into symmetrical spindle-shaped cells (Figure 1c Cells were treated with Src Inhibitor No. 5, a selective inhibitor of Src tyrosine kinases, to investigate its influence on the physiological characteristics of fibroblasts and elucidate the mechanisms underlying polarized cell elongation. Normal fibroblasts cultured in medium containing 10 µM Src Inhibitor No. 5 first adhered to the glass substrate and then showed a symmetrical spindle-like extension (Figure 2, arrowheads), similar to the morphology of SYF cells stretched on the glass substrate (see Figure 1c), as shown in the time-lapse phase-contrast microscopy images (Video S1). Cells were treated with Src Inhibitor No. 5, a selective inhibitor of Src tyrosine kinases, to investigate its influence on the physiological characteristics of fibroblasts and elucidate the mechanisms underlying polarized cell elongation. Normal fibroblasts cultured in medium containing 10 μM Src Inhibitor No. 5 first adhered to the glass substrate and then showed a symmetrical spindle-like extension ( Figure 2, arrowheads), similar to the morphology of SYF cells stretched on the glass substrate (see Figure 1c), as shown in the time-lapse phase-contrast microscopy images (Video S1). After washing the c-Src inhibitor-treated cells (shown in Figure 2) with an inhibitor-free culture medium, the symmetrically stretched fibroblasts began to show directed migration and extended pseudopodia and eventually exhibited typical fibroblast morphology ( Figure 3; see also Video S2).
Next, we examined the effects of transfection of cultured fibroblasts with a dominant negative Src expression vector (Figures 4 and 5). Mutation of Lys295 in the catalytic site to methionine (K295M) inactivates Src kinase activity [43,44]; studies on mutant cells have indicated that both splicing and transport activities require the kinase activity of Src [45]. An Src mutant containing both Src K295M/Y527F mutations was also generated and was shown to have an open conformation and no kinase activity but to retain SH2 and SH3 binding activity due to the lack of interaction with intramolecular SH2-pTyr527 [46,47]. A constitutively active Src mutant was also generated by mutating the inhibitory Tyr527 to phenylalanine (Y527F). The dominant negative c-Src gene (pUSEamp-dominant negative c-Src) was expressed in normal fibroblasts, and living cells were recorded under phasecontrast microscopy ( Figure 4). Cells expressing the dominant negative c-Src gene were elongated at both poles in a symmetrical cone shape.  After washing the c-Src inhibitor-treated cells (shown in Figure 2) with an inhibit free culture medium, the symmetrically stretched fibroblasts began to show directed m gration and extended pseudopodia and eventually exhibited typical fibroblast morph ogy (Figure 3; see also Video S2).  Next, we examined the effects of transfection of cultured fibroblasts with a dominant negative Src expression vector (Figures 4 and 5). Mutation of Lys295 in the catalytic site to methionine (K295M) inactivates Src kinase activity [43,44]; studies on mutant cells have indicated that both splicing and transport activities require the kinase activity of Src [45]. An Src mutant containing both Src K295M/Y527F mutations was also generated and was shown to have an open conformation and no kinase activity but to retain SH2 and SH3    To examine c-Src expression in the polarization of cells, we transfected the constitutively active or dominant negative form of c-Src into normal fibroblasts ( Figure 5). Here, pUSEamp-WT c-Src (Figure 5a,b), pUSEamp-constitutively active c-Src (Y527F; Figure  5d,e), and pUSEamp-dominant negative c-Src (K295M/Y527F; Figure 5d,e for a single image of the fixed cells) were transfected into normal fibroblasts. Some cells were stained with an antibody to vinculin as a marker of focal adhesions (Figure 5b,d,f). The cells expressing WT c-Src showed almost the same morphology as normal fibroblasts ( Figure  5a  To examine c-Src expression in the polarization of cells, we transfected the constitutively active or dominant negative form of c-Src into normal fibroblasts ( Figure 5). Here, pUSEamp-WT c-Src (Figure 5a,b), pUSEamp-constitutively active c-Src (Y527F; Figure 5d,e), and pUSEamp-dominant negative c-Src (K295M/Y527F; Figure 5d,e for a single image of the fixed cells) were transfected into normal fibroblasts. Some cells were stained with an antibody to vinculin as a marker of focal adhesions (Figure 5b,d,f). The cells expressing WT c-Src showed almost the same morphology as normal fibroblasts (Figure 5a Figure 5f, staining for vinculin). The cells expressing constitutively active c-Src showed a pancake-like morphology (Figure 5c), and vinculin-positive focal adhesions were observed mainly at the cell periphery but not in the center of the cell (Figure 5d). SYF cells expressing eGFP-fused WT c-Src (Figure 6a) and eGFP-fused dominant negative c-Src (Figure 6b) were examined by phase-contrast microscopy. Transfection of the cells with each eGFP-fusion gene construct was examined by fluorescence microscopy, and photographs were taken under phase-contrast microscopy. Cells expressing eGFP-fused dominant negative c-Src had a symmetrical shape (Figure 6b). SYF cells cultured on a glass substrate were extended with a symmetrical slim spindle-like structure. When transfected with a vector carrying the wild-type c-Src gene, SYF cells were extended with pseudopodia and showed normal fibroblast-like morphology.
staining for vinculin). The cells expressing constitutively active c-Src showed a pancakelike morphology (Figure 5c), and vinculin-positive focal adhesions were observed mainly at the cell periphery but not in the center of the cell (Figure 5d).
SYF cells expressing eGFP-fused WT c-Src (Figure 6a) and eGFP-fused dominant negative c-Src (Figure 6b) were examined by phase-contrast microscopy. Transfection of the cells with each eGFP-fusion gene construct was examined by fluorescence microscopy, and photographs were taken under phase-contrast microscopy. Cells expressing eGFPfused dominant negative c-Src had a symmetrical shape (Figure 6b). SYF cells cultured on a glass substrate were extended with a symmetrical slim spindle-like structure. When transfected with a vector carrying the wild-type c-Src gene, SYF cells were extended with pseudopodia and showed normal fibroblast-like morphology. Normal fibroblasts transfected with a vector encoding dominant negative c-Src adhered to the glass substrate and began to extend in both polar regions, eventually becoming spindle-shaped cells. The mean ± standard error of the mean (SEM) aspect ratio of the vertical axis to the long axis of the SYF cells transfected with dominant negative c-Src was 7.51 ± 0.43 (n = 50). The aspect ratio of SYF cells was 5.98 ± 0.24, while the aspect ratio of normal fibroblasts was 2.38 ± 0.13 (Table 1). SYF cells cultured on a glass substrate adopted a symmetrical spindle shape. SYF cells transfected with a vector carrying WT c-Src became elongated, had extended pseudopodia, and showed almost the same morphology as normal fibroblasts. Normal fibroblasts transfected with the vector carrying the dominant negative c-Src gene adopted a symmetrical spindle shape. On treatment with the specific c-Src inhibitor, Src Inhibitor No. 5, normal fibroblasts were elongated in a similar manner to SYF cells, with focal adhesions formed at both ends of the elongated cells. Following washout of the c-Src inhibitor, the pseudopodia were actively extended, and the cells returned to a morphology that was almost identical to normal fibroblasts. These observations suggest that polarity formation during cell extension and elongation is regulated by the SFKs and that the expression and regulation of at least one member of the family, c-Src, are important for polarity formation during cell elongation.

Discussion
Signal transduction mechanisms in cells involve tyrosine phosphorylation for activation or inactivation of specific proteins. The levels of tyrosine phosphorylation reflect the local levels of signal transduction activity. Phosphotyrosine proteins are highly accumulated at the sites of focal adhesion in cells in culture, reflecting the involvement of these sites in signal transduction. SFKs are membrane-bound, nonreceptor tyrosine kinases that function as important signaling intermediates in the regulation of cell proliferation, differentiation, apoptosis, migration, and metabolism [21,25,48].
Due to their role in regulating cellular adhesion, the turnover of integrins by endocytosis or exocytosis is necessary for cell movement [49]. These processes seem to be controlled by FAK and its substrates [50], including SFKs [51]. SFK is a family of oncogenes that were initially discovered in association with cancer. Tumors in chickens were shown to be caused by the Rous sarcoma virus oncogene, v-Src, which is similar to the typical cellular protein, c-Src. Unlike c-Src, v-Src is constitutively active as it lacks the C-terminal inhibitory phosphorylation site (Y527) [52]. The c-Src protein is a signaling molecule that has important roles in controlling cell growth, proliferation, and/or motility.
Stress fibers are a contractile apparatus that can generate isometric tension in cells, which is possible because both ends of the stress fibers are anchored to the substrate via focal adhesions [17]. Cell motility seems to be regulated by the crosstalk between Rho, Rac, Ras, and/or Cdc42. Although the mechanisms underlying the regulation of cell polarity are still unknown, the results of the present study indicate the roles of activation and deactivation of c-Src in the control of cell polarity. Normal fibroblasts transfected with dominant negative c-Src showed greater cell elongation than control cells.
In this study, SYF cells transfected with dominant negative c-Src retained an elongated morphology. In addition, WT c-Src-transfected SYF cells showed almost the same morphology as normal fibroblasts. When the cell migrates, the leading edge shows a filopodium-like structure. Migrating cultured cells have polarity, with a leading edge at the front of the cell and a trailing edge at the rear of the cell. However, the mechanism by which the leading edge and the trailing edge are specified is still unclear. The results of the present study suggest that deactivation of c-Src results in an elongated morphology and a symmetrical cell shape [19].
SFKs are translocated to the sites of cell adhesion [53]. Previous studies have shown that Src kinase activity influences cell proliferation and cell migration [54][55][56]. Moreover, the results presented here suggest that the kinase activity of SFKs plays a role in the regulation of symmetrical elongation of fibroblastic cells under physiological conditions. SYF cells showed no consistent differences in the actin cytoskeleton in comparison to WT controls. Focal adhesions form in the absence of tyrosine phosphorylation of focal adhesion-associated proteins, and SYF cells show reduced motility [34]. In this study, the reintroduction of WT c-Src into SYF cells restored the defective motility and bipolarity of the fibroblastic cells. SYF cells formed focal adhesions, which showed a typical arrowhead-like appearance and were present in the same numbers as in normal fibroblasts; no differences in the number of stress fibers were observed [57]. The present study demonstrates that SYF cells and normal fibroblasts transfected with dominant negative c-Src will show reduced cell motility, consistent with previous reports. Moreover, fibroblasts transfected with dominant negative c-Src showed polarized movement, with a symmetrical cell shape. These results indicate that the deactivation of c-Src plays a role in cell motility, resulting in cell polarization. With regard to fibroblastic cell proliferation, the activity of Src is physiologically important to the polarized elongation of the cell.
The initiation of Src tyrosine kinase signal transduction pathways leads to the proliferation and activation of fibroblasts, which deposit extracellular matrix into the surrounding connective tissue. Fibrosis, defined by the accumulation of excess extracellular matrix components, is a pathological feature of most chronic inflammatory diseases. The results of the present study show that Src tyrosine kinase is a candidate molecule involved in the regulation of fibroblast elongation and polarization. The interaction of Src with integrin αV is required for integrin αV-mediated Src activation and subsequent fibroblast migration [58]. The interaction of SFKs and integrins plays critical roles in the development of lung fibrosis [59][60][61], liver fibrosis [62,63], and chronic kidney disease [64]. SFKs seem to play critical roles in the development of lung fibrosis, and the signaling involved may represent a novel opportunity to target fibrotic diseases [58]. The pathogenesis of these diseases is not fully understood, but they do appear to be associated with alterations in fibroblast migration and excessive matrix deposition [65,66]. Fibroblast migration and proliferation are tightly controlled processes [67,68]. Protein-kinase-regulated cell migration is involved in the development of lung fibrosis [66]. Src tyrosine kinase regulates focal adhesion kinase activation and seems to cause cell elongation [69]. However, the involvements of Src tyrosine kinase in fibroblast migration and proliferation and in lung fibrosis have yet to be explored in detail. Treatment with the Src inhibitor, PP2, was reported to significantly reduce fibroblast migration stimulated by platelet-derived growth factor-BB (PDGF-BB) and to reduce lung fibrosis in mice in vivo [58]. The results of the present study indicate that c-Src tyrosine kinase is involved in fibroblastic polarized cell elongation and symmetrical cell shape changes. It has been suggested that Src tyrosine kinases are involved in the pathogenesis of renal fibrosis, and the selective Src tyrosinase inhibitor PP1 may inhibit fibrosis and have therapeutic potential for the treatment of CKD chronic kidney disease [70]. The findings outlined above suggest that SFKs play roles in the development of fibrosis that is related to the proliferation and elongation of fibroblasts.
The mechanism by which the cell determines the direction of migration is still unclear. The direction of migration seems to be determined randomly, according to the orientation of stress fibers in cultured cells. The position of the Golgi apparatus appears to be critical in specifying various aspects of cell migration [71]. Disruption of the Golgi apparatus, even under conditions where the cytoskeleton remains intact, results in failure of cell polarization and inhibition of cell migration [71]. The microtubule organization center (MTOC) and its associated microtubules determine the position of the Golgi apparatus [72], which is thought to facilitate polarized secretion of the plasma membrane at the leading edge of the cell [73]. The positions of the Golgi apparatus and MTOC have been suggested to be involved in determining the direction of cell migration. However, as focal adhesions represent the footholds of migrating cells, elongation and determination of polarization seem to be determined to a greater extent by cytoskeletal components than the Golgi and/or MTOC associated with focal adhesions.
In this study, cells transfected with a dominant negative c-Src construct showed a symmetrical shape, suggesting that the deactivation of c-Src causes bidirectional elongation. Moreover, the formation of pseudopodia at the leading edge of the cell was reduced. Well-developed focal adhesions were detected at both ends of the leading edge of these cells. Random migration was inhibited in cells transfected with the dominant negative c-Src construct. The Arp 2/3 complex is involved in the regulation of the actin-containing cytoskeleton in pseudopodia at the leading edge of the cell. Arp 2/3 interacts with the small rho-type GTPase Cdc42, and, thus, Arp 2/3 may align actin filaments to the plasma membrane through the activity of Cdc42 [3,74]. Deactivation of c-Src resulted in a lack of pseudopodia at the leading edge of the cell (Figure 5), reflecting the disorganization of pseudopodia in both c-Src-deficient cells and cells with deactivated c-Src. The above observations suggest that the deactivation of c-Src also causes Cdc42-dependent organization of the Arp 2/3 meshwork in pseudopodia structures.
SYF cells lacking the SFK members Src, Yes, and Fyn, cultured on glass substrates, were observed to elongate into symmetrical spindle-shaped cells. The introduction of the WT c-Src gene into SYF cells resulted in the elongation and extension of pseudopodia, and they adopted a morphology that was similar to normal fibroblasts. Normal fibroblasts transfected with a construct carrying the dominant negative c-Src gene adopted a symmetrical spindle shape. Treatment of normal fibroblasts with the c-Src-specific inhibitor, Src Inhibitor No. 5, resulted in symmetrically shaped cells, similar in morphology to SYF cells. Focal adhesions were formed at both ends of the elongated cells. Following washout of the c-Src inhibitor, the pseudopodia were actively extended, and the cells returned to a morphology that was almost identical to normal fibroblasts. Other Src family inhibitors caused the cells to adhere to the glass substrate but not to adopt the spindle-shaped morphology. These observations address the question of how the symmetrically elongated features of the cell are organized. The results of the present study indicate that cells transfected with the dominant negative c-Src gene or incubated with a c-Src inhibitor will first attach to the glass substrate with a pancake-like morphology, after which they elongate along both poles and adopt a symmetrical shape within 2 h. Inactivation of c-Src results in highly elongated cells, both ends of which are slender and narrow in shape.
The results of the present study suggest that polarity formation during cell extension and elongation may be regulated by the Src family and that the expression and regulation of at least one member of the family, c-Src, are important for polarity formation during cell elongation.
Funding: The work reported here was supported by Grants-in-Aid for Promotional Projects for Advanced Education and Research, National University, in cooperation with Tsukuba University of Technology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.