The major findings of this study comparing the location and function of the three large tensin isoforms are as follows: (1) Each tensin isoform has a distinctive subcellular location. Tensin 2 is present at the front edge of growing focal adhesions at the leading edge of primary human fibroblasts; its levels are dynamic but it does not translocate along adhesions; tensin 1 and tensin 3 are present in fibrillar adhesions further back from the leading edge. However, tensin 1 remains stable and higher in anterior adhesions, while tensin 3 translocates back along adhesions and is enriched in fibrillar adhesions under the cell body. (2) Contraction of 3D collagen gels by human fibroblasts is dependent on tensin 2 through modulation of Rho activity, and it does not depend on tensins 1 or 3. (3) Depletion of the RhoGAP DLC1, which binds to tensin in focal adhesions, reverses the inhibition of collagen gel contraction after tensin 2 knockdown.
Although some previous work has attempted to localize the tensin isoforms in different types of cell adhesions, no direct comparisons of localization of the various isoforms has been performed in the same cell type. In NIH 3T3 cells, GFP-tensin 2 localized to focal adhesions and did not target fibrillar adhesions as efficiently as tensin 1 [Chen et al., 2002
]. Tensin 3 was found in focal adhesions of A549 lung epithelial cells, but these cells are unlikely to make fibrillar adhesions and were used because of antibody cross-reactivity problems with tensin 1 [Cui et al., 2004
]. We clarified by determining the antibody specificity that the rat tensin localized in both focal and fibrillar adhesions in REF52 cells [Zamir et al., 1999
] is tensin1. Extending these results, we find that there is a hierarchy of tensin localization to cell adhesions, with tensin 2 being closest to the leading edge followed by tensin 1, and then tensin 3. The anterior location and dynamics of tensin 2 at sites ideally suited for early interactions with extracellular matrix suggest a role in matrix remodeling. In contrast, tensin 3 which has the ability to fully translocate centripetally as the adhesion grows is predominantly localized in more posterior fibrillar adhesions. Previous live cell imaging using GFP-chicken tensin also indicated translocation of tensin into longer fibrillar adhesion-like structures [Zamir et al., 2000
]. The highly conserved nature of the N- and C-terminal regions of these proteins suggests that it is the central divergent and apparently unstructured regions of the tensin isoforms that are likely involved in their differing cellular localizations and functions.
Although tensins are a major cytoskeletal component of fibrillar adhesions (Zamir et al., 1999
), and expression of a chicken tensin fragment, the AH2 region, blocked the formation of fibrillar adhesions and the process of fibronectin fibrillogenesis (Pankov et al., 2000
), knockdown of all three tensin isoforms had no apparent effect on the assembly of fibronectin matrix under the conditions we used. It is not understood how the AH2 region mediates its dominant-negative effect on fibrillar adhesion formation. The sequence of this region of chicken tensin is not conserved within the human tensin isoforms, but it falls within the unstructured central region and may function by coupled folding and binding to its functional partners [Wright and Dyson, 2009
]. The tensin family of proteins may not be essential for fibronectin fibrillogenesis, or compensation by other linker proteins such as talin, ILK or PINCH may occur [Feral et al., 2007
; Green et al., 2009
; Guo and Wu, 2002
; Shi et al., 2008
; Wu et al., 1998
]. Indeed, a recent study has shown that ILK and PINCH1 are essential for focal adhesion maturation into fibrillar adhesions, and tensin availability did not alter the fibrillar adhesion defect in the ILK or PINCH knock-out cells. However, ILK and α-parvin are required for tensin recruitment to fibrillar adhesions [Stanchi et al., 2009
]. Since tensin 2 is a dynamic component of focal adhesions, we therefore investigated its role in remodeling of a 3D ECM.
Collagen gel contraction has been used as a model for pro-contractile remodeling of the ECM by fibroblasts in processes such as wound healing [Grinnell, 2003
]. The collagen gel system we used allows cells to develop endogenous tension strong enough to separate the gel from the dish edge. This method depends on signaling mechanisms important for cell contractility [Grinnell, 2000
]. Using this system, we showed that HFF gel contraction is dependent on β1 integrin, most probably the two major collagen receptors α2β1 and α11β1 integrin [Carver et al., 1995
; Cooke et al., 2000
] and is inhibited by very low doses of the Rho inhibitor C3 exotransferase, indicating that it is highly sensitive to the global level of Rho activity. Therefore, it was particularly significant to find that knockdown of tensin 2 expression substantially inhibited the activity of Rho and impaired the ability of the cells to contract the collagen gel. Depletion of tensin 1 or 3 had no effect on collagen gel contraction. Interestingly transfection of MDA-MB 231 breast cancer cells with tensin 1 mutants F302A or R1488A, which bind reduced levels of DLC1, have 2-fold lower levels of active Rho compared to wild-type tensin 1 [Hall et al., 2009
]. In contrast, fibroblasts taken from plastic surgery and transfected with high concentration of tensin 1 siRNA demonstrated a reduced collagen gel contraction, this correlated with aged fibroblasts having reduced tensin 1 expression and capability to contract collagen gels [Saintigny et al., 2008
]. The higher concentration of siRNA used in this study may cause off target effects, and the source and passage number of the fibroblasts may also influence the experimental outcomes.
Although depletion of all tensin isoforms affected Rho activation levels, tensin 2 knockdown had the greatest effect, reducing Rho activity by 80%. The RhoGAP DLC1 has been shown to localize to focal adhesions [Kawai et al., 2004
] and was subsequently identified as a tensin-binding protein [Yam et al., 2006
]. DLC1 interacts with the SH2 domain of all tensins in a tyrosine phosphorylation-independent manner, via Tyr442, which is essential for DLC1 localization to the focal adhesion [Liao et al., 2007
; Qian et al., 2007
]. DLC1 has been found subsequently to have an essential binding site for the tensin 2 PTB domain, which may be involved in tensin 2-specific recruitment of DLC1 to the focal adhesion [Chan et al., 2009
]. DLC1 has been identified as a tumor suppressor and is an inhibitor of cell growth. However, because the collagen gel contraction assays were performed in relaxed gels where the cells are quiescent, the ability of DLC1 to suppress cell growth is unlikely to have affected the outcome of the results reported here. In addition, no difference in cell viability was observed between the different siRNA-transfected cells. We therefore speculated that experimental depletion of tensin 2 might result in unregulated DLC1 activity at the focal adhesion, and its RhoGAP function would reduce Rho activity in a specific localized manner and limit gel contraction. Consistent with this hypothesis, knockdown of DLC1 dramatically rescued the ability of fibroblasts depleted of tensin 2 to contract collagen gels. The DLC1 knock-down alone had no effect on gel contraction, indicating a tensin2 regulatory role. DLC1 RhoGAP activity can be inhibited by intramolecular interaction through its SAM domain [Kim et al., 2008
] or by the more recently discovered interaction with p120Ras-GAP, a DLC1 inhibitor [Yang et al., 2009
]. p120Ras-GAP co-localizes with DLC1 at the focal adhesion, where tensin 2 is also present. The recently identified binding site for the tensin 2 PTB domain within DLC1 [Chan et al., 2009
] indicates a different relationship between these proteins to that between DLC1 and the other tensin isoforms. This leads us to suggest that tensin 2 inhibits DLC1 either by stabilizing the intramolecular interaction with the SAM domain or promoting the interaction between DLC1 and p120Ras-GAP. Loss of tensin 2 could therefore reduce Rho activity and contractility by permitting activation of the DLC1 RhoGAP.
In conclusion, we have demonstrated distinct localization and dynamics of all three large tensin isoforms. Tensin 2 is highly dynamic and is enriched in focal adhesions, whereas tensin 3 mainly locates to fibrillar adhesions, and tensin 1 appears to be equally distributed between both adhesion types. Tensin 2 plays a critical role in regulating Rho activity and collagen gel contraction by a novel mechanism in which it locally modulates DLC1 RhoGAP function in focal adhesions at the leading edge, permitting Rho activation and subsequent matrix remodeling.