Progesterone enhances vascular endothelial cell migration via activation of focal adhesion kinase

Abstract The mechanisms of progesterone on endothelial cell motility are poorly investigated. Previously we showed that progesterone stimulated endothelial cell migration via the activation of actin-binding protein moesin, leading to actin cytoskeleton remodelling and the formation of cell membrane structures required for cell movement. In this study, we investigated the effects of progesterone on the formation of focal adhesion complexes, which provide anchoring sites for cell movement. In cultured human umbilical endothelial cells, progesterone enhanced focal adhesion kinase (FAK) phosphorylation at Tyr397 in a dose- and time-dependent manner. Several signalling inhibitors interfered with progesterone-induced FAK activation, including progesterone receptor (PR) antagonist ORG 31710, specific c-Src kinase inhibitor PP2, phosphatidylinosital-3 kinase (PI3K) inhibitor wortmannin as well as ρ-associated kinase (ROCK-2) inhibitor Y27632. It suggested that PR, c-Src, PI3K and ROCK-2 are implicated in this action. In line with this, we found that progesterone rapidly promoted c-Src/PI3K/Akt activity, which activated the small GTPase RhoA/ρ-associated kinase (ROCK-2) complex, resulting in FAK phosphorylation. In the presence of progesterone, endothelial cells displayed enhanced horizontal migration, which was reversed by small interfering RNAs abrogating FAK expression. In conclusion, progesterone promotes endothelial cell movement via the rapid regulation of FAK. These findings provide new information on the biological actions of progesterone on human endothelial cells that are relevant for vascular function.


Introduction
The endothelium plays a primary role in the regulation of vascular structure and functions. Endothelial dysfunction is a well-established response to cardiovascular risk factors and can be a promoter of atherosclerosis [1,2]. Menopause is associated with endothelial dysfunction because of sex steroids reduction. In this regard, the impaired flow-mediated dilatation, reduced nitric oxide availability and abnormal endothelial morphology are found in post-menopausal women [3][4][5]. These functional abnormities may explain the increased incidence of cardiovascular disease in post-menopausal women.
Hormone replacement therapy (HRT) is believed to induce cardiovascular protection. However, clinical trials have challenged this assumption, reporting no cardiovascular benefit or even increased risk in post-menopausal women receiving HRT [6][7][8][9][10]. This apparent discrepancy is still under investigation. Recent years a 'healthy endothelium' concept has been proposed, suggesting that the cardiovascular effects of HRT may largely depend on intact endothelial function [11]. In this regard, the Cardiovascular Health Study demonstrated that favourable vascular effects of HRT are only limited to patients without extensive atherosclerosis [3]. In parallel, our previous work indicated that HRT improves the vascular function and preserves the morphological integrity of endothelial cells in relatively young post-menopausal women [12].
Endothelial cells are primary targets of sex steroids. It is well recognized that oestrogen improves endothelial function through genomic and non-genomic mechanisms [13]. By contrast, less is known on the progesterone's endothelial actions. We recently 297 discovered that progesterone stimulates endothelial migration [14]. By promoting dynamic actin remodelling, progesterone activates the actin-regulatory protein moesin, which supports the formation of membrane filopodia and pseudopodia [14]. These structures are essential for cell migration by interaction with the extracellular matrix via anchorage proteins and focal adhesions (FAs), which provide the platform for cells to generate the locomotive force. Nevertheless, it remains largely unknown whether progesterone regulates endothelial cell adhesion to the extracellular matrix.
FAs are composed of various structural proteins and represent sites where a number of intra-and extracellular signalling events regulating cell migration take place. Focal  in the catalytic loop, which is necessary for the full activation of the kinase domain. Activated FAK begins to partner with cellmembrane integrins with the assistance of other proteins such as paxillin and vinculin, resulting in FA formation and cell migration [15]. FAK activity is essential not only for tumour metastasis [16], but also for developmental processes controlling blood vessel formation [17].
Recently we found that progesterone modulates FAK activity in breast cancer cells [18]. In this study, we suggested that FAK is the target of progesterone in vascular endothelial cells and its activation plays important role in progesterone-stimulated endothelial cell migration. Therefore, we explored the regulatory actions of progesterone on FAK activity by using Western blot and immunofluorescence methods. The role of active FAK on endothelial migration was analysed using the wound healing assay. Moreover, by transfection with specific small interference RNA (siRNA) or overexpression plasmids, we characterized the signalling pathways initiated by progesterone receptor (PR) that lead to FAK activation.

Cell cultures and treatments
Human umbilical vein endothelial cells (HUVEC) were cultured as previously described [19]. Before treatments, HUVEC were kept 48

Cell migration assays
Cell migration was assayed with razor scrape assays as previously described [19]. Briefly, a razor blade was pressed through the confluent HUVEC monolayer into the plastic plate to mark the starting line. HUVEC were swept away on one side of that line. Cells were washed, and 2.0 ml of DMEM containing steroid-deprived FBS and gelatine (1 mg/ml) were added. Cytosine ␤-D-arabinofuranoside hydrochloride (Ara-C, Sigma) (10 M), a selective inhibitor of DNA synthesis, which does not inhibit RNA synthesis was used 1 hr before the test substance was added. Ara-C is easy to enter into the cellular nucleus and it incorporates into DNA and inhibits DNA replication by the formation of cleavage complexes with topoisomerase I resulting in DNA fragmentation. Although the half-life of Ara-C was less than 1 hr in most of the cell lines, more than 80% of Ara-C DNA was retained at 24 hrs after drug removal [20]. Migration was monitored for 48 hrs. Fresh medium and treatment were replaced every 12 hrs. Cells were digitally imaged and migration distance was measured by using phase-contrast microscopy.

Statistical analysis
All values are expressed as mean Ϯ S.D. Statistical differences between mean values were determined by Turkey-Kramer test for multiple comparisons. All differences were considered significant at P Ͻ 0.05.

Progesterone activates FAK and increases the formation of focal adhesion complexes
First HUVEC was treated with progesterone in a range of concentrations (10 Ϫ10 -10 Ϫ7 mol/l). It is based on the fact that the serum level of progesterone in female is 1 to 90 ng/ml, which nearly equals to 0.3 to 300 nmol/l [21]. As shown in Figure 1A, progesterone rapidly induced FAK phosphorylation at Tyr 397 (which corresponds to activation [15]) and Tyr 576/577 (which is necessary for the full activation of kinase domain [22]). Tyr 397 of FAK was phosphorylated by progesterone at 10 Ϫ8 mol/l with a visible effect from 5 min., a peak at 30 min. and a decline to basal levels after 1 hr (Fig. 1B).

Meanwhile, the sub-cellular localization of phosphorylated FAK in endothelial cells was examined by immunofluorescent analysis.
In vehicle-treated control cells, the immunostaining of phosphorylated FAK was rather weak and actin fibres were longitudinally arranged in the cytoplasm (Fig. 1C). Short-term exposure to progesterone (10 Ϫ8 mol/l) led to increased staining of phosphorylated FAK, which concentrated at the cell membrane where actin remodelling happened. These alterations could be observed as early as 5 min. and began to revert to baseline after 1 hr (Fig. 1C).

c-Src and phosphatidylinositol-3 kinase (PI3K)/Akt are required for FAK activation
The rapid time-lapse of FAK activation and de-activation suggests that this effect is likely to be extra-nuclear [23]. Indeed, progesterone still led to FAK activation even if RNA or protein synthesis was blocked in HUVEC with Act D (10 M) or CHX (200 M) ( Fig. 2A). In addition, FAK activation was also induced by the membrane-impermeable albumin-progesterone conjugate (PBSA, 10 nM) ( Fig. 2A).
To clarify the signalling molecules responsible for progesterone-induced FAK activation, we interfered with some of the cascades that mediate extra-nuclear actions of progesterone. Blockade of PR with the pure PR antagonist ORG 31170 (1 M) completely abolished progesterone-dependent FAK activation, suggesting that PR is indispensable for progesterone's effect (Fig. 2B). Meanwhile, PI3K inhibitor WM (30 nM), non-receptor tyrosine kinase c-Src inhibitor PP2 (10 M) and ROCK-2 Y27632 (10 M) all largely impaired progesterone-induced FAK activation (Fig. 2B), indicating that c-Src, PI3K and ROCK-2 are required for this action.
On the contrary, progesterone-enhanced FAK phosphorylation was not altered by the G protein inhibitor PTX (100 ng/ml) or the mitogen-activated protein kinase kinase inhibitor PD98059 (5 M) (Fig. 2B).
PR interacts with the tyrosine kinase c-Src [24] and this process is involved in the activation of PI3K [25]. In line with this, progesterone (10 Ϫ8  corresponds to its activation [26] (Fig. 2C). Likewise, the PI3K downstream effector, protein kinase Akt, was also phosphorylated in response to progesterone (Fig. 2C). Moreover, the PI3K inhibitor WM had no effect on c-Src activation, whereas the c-Src inhibitor PP2 blocked Akt phosphorylation (Fig. 2C), indicating that c-Src was the upstream of Akt in this experimental setting.
The functional importance of c-Src/PI3K/Akt cascade in FAK activation was further elucidated by the transfection experiments.  Progesterone was unable to activate Akt when c-Src expression was silenced with specific siRNA (Fig. 2D). In addition, transfection with a dominant-negative form of the regulatory subunit of PI3K, p85␣ (⌬p85␣), resulted in the impairment of FAK activation, whereas the transfection of a wild-type p85␣ construct (WT p85␣) showed an additive effect with progesterone (Fig. 2E). The p85 subunit were overexpressed in both WT p85␣ and ⌬p85␣ plasmid transfection group (Fig. 2E), implying an efficient transfection in our experimental settings. Taken together, these results suggest that the c-Src/PI3K/Akt cascade is implicated in the PR-dependent FAK activation.

Progesterone-induced FAK activation: role of RhoA/ROCK-2
The small GTPase RhoA and its downstream effector ROCK-2 function as the modulators of actin cytoskeleton by linking upstream signalling events to actin-binding proteins such as moesin or FAK [27,28]. Recently we have shown that progesterone enhanced RhoA/ROCK-2 activities in breast cancer cells [18]. Similarly, treatment of HUVEC with progesterone increased the amount of active, GTP-bound RhoA (Fig. 3A) and of functionally activated ROCK-2, shown by enhanced Thr-phosphorylation of the bait protein MBP by ROCK-2 immunoprecipitates (IPs) (Fig. 3B).
The activation of RhoA/ROCK-2 is mediated by c-Src and PI3K, because inhibitors of both kinases blocked their activation induced by progesterone (Fig. 3A, B). In line with this, RhoA activation was impaired by transfection with ⌬p85␣, whereas transfection with WT p85␣ increased RhoA activation in the presence or absence of progesterone (Fig. 3C).
Y27632, the inhibitor of ROCK-2, was shown to largely impair progesterone-induced FAK activation (Fig. 2B) (30 nM), of PP2 (10 M). ROCK-2 was immunoprecipitated with a specific Ab and the IPs were used to phosphorylate the bait protein, MBP. ROCK-2 kinase activity is shown as the amount of phosphorylated MBP (P-MBP). P-MBP densitometry values were adjusted to ROCK-2 intensity, then normalized to expression from the control sample. **P Ͻ 0.01 versus control. # P Ͻ 0.01 versus P. (C) HUVECs were exposed to P (10 nM) for 15 min. after transfection with wildtype p85␣ (WT p85␣) or dominant-negative p85␣ (⌬p85␣) for 48 hrs. Active, GTP-bound RhoA was assayed and total RhoA content was analysed in the input. RhoA-GTP densitometry values were adjusted to total RhoA intensity, then normalized to expression from the control sample. **P Ͻ 0.01 versus control without transfection. (D) HUVECs were either mock-transfected or exposed to constitutively active or dominant-negative RhoA (RhoA CA or RhoA DN). Cells were then treated with P (10 nM) for 15 min. and wild-type or phosphorylated FAK were analysed. pFAK and RhoA densitometry values were adjusted to FAK intensity, then normalized to expression from the corresponding control sample. **P Ͻ 0.01 versus corresponding control without transfection. # P Ͻ 0.01 versus P. All these experiments were performed in triplicates and representative images are shown.

FAK activation is critical for HUVEC migration
Finally the role of FAK activation in endothelial cell migration was interrogated. Consistent with our previous work [14], progesterone markedly enhanced horizontal migration (Table 1). This effect was completely blocked by silencing FAK with siRNAs. The enhancement of endothelial migration induced by progesterone was also prevented by blocking PR with ORG 31710, by blocking c-Src with PP2, PI3K with WM, ROCK-2 with Y-27632 (Table 2). [13]. However, little is known about the progesterone's effects on endothelial cells. The major finding of this proposal is the identification of endothelial FAK as the target of progesterone that promotes endothelial cell migration.

Female sex hormones have vascular benefits and this is largely attributed to their direct actions on vascular endothelial cells. It is well documented that oestrogen is the potent agent to maintain endothelial morphology and functions
Endothelial migration is critical for the physiological or pathophysiological processes such as vessel repair, angiogenesis and wound repair. Cell migration is a highly integrated process implemented by actin reorganization. Our previous studies showed that progesterone induces dynamic actin remodelling and the formation of membrane filopodia and pseudopodia, which are primary steps for endothelial cell migration [14]. This event is linked to the rapid activation by PRs of actin-binding protein moesin, which belongs to the ezrin/radixin/moesin family [29]. Protruded mem-branes then contact the substrate and form novel integrin-dependent FAs, which provides the platform for cells to generate the locomotive force [30]. Besides its ability to provoke actin cytoskeleton remodelling, progesterone is also revealed to increase the formation of FAs, indicating that progesterone may impact on endothelial migration through the modulation of multiple migratory steps.
We here find that progesterone rapidly activates Tyr 397 phosphorylation of endothelial FAK, leading to the formation of FA complexes. Endothelial FAK is essential for vascular morphogenesis and vascular repair due to its central role on endothelial cell migration [31,32]. For example, in the transgenic mice which overexpressed FAK in vascular endothelial cells, the number of vessels in the granulation tissue of healing wound is significantly increased [33]. Likewise, the inhibition of FAK phosphorylation results in reduced endothelial repair in cell wounding model [34]. These findings highlight the relevance of the active endothelial FAK in the maintenance of vascular functions and integrity. The identification of endothelial FAK regulation by progesterone may thus offer important mechanistic insights to better understand the beneficial cardiovascular effects of this natural hormone [35][36][37].
The phosphorylation of FAK is characterized by the rapid time lapse and is insensitive to RNA and protein synthesis inhibitors, indicating that this action induced by progesterone is achieved via extra-nuclear signalling cascades. Although the definition of the exact membrane-binding site for progesterone is beyond the scope of this article, there is a possibility that signalling responsible for FAK activation are initiated from liganded-membrane PRs. Indeed, the existence of membrane-localized PRs in different tissues (including vascular endothelial cells) is well established [38][39][40]. Whatever the sub-cellular localization of PRs that are required to start signalling to endothelial FAK, the present findings concur with our previous reports in suggesting that membrane    [19,28,41]. Although the present study displays a tight relationship between FAK activation and endothelial cell motility, the overall contribution of this rapid action to the long-term migration remains to be clarified. Indeed, progesterone may also alter FAK activity through conventional nuclear actions [42]. For instance, long-term treatment with progesterone or synthetic progestin medroxyprogesterone acetate (MPA) increases the expression and phosphorylation of FAK and the formation of FA complexes, resulting in enhanced migration of vascular endothelial cells or of breast cancer cells [12,43].
A critical step for full enzymatic activity of FAK is the Tyr 397 phosphorylation in the kinase domain, followed by the phosphorylation of Tyr 576/577 in the catalytic loop [44,45]. A variety of stimuli, such as mechanical stress or the activation of growth factor receptors, catalyse Tyr 397 phosphorylation and the active FAK functions as a molecular switch for multiple signalling outputs [46,47]. In this cascade of events, c-Src acts as a central hub relaying upstream signals to FAK as well as conveying signals from FAK to downstream effectors [15]. It has been reported that PR contains a proline-rich motif that directly interacts with SH3 domains and activates c-Src [24]. In line with this, the present study finds that progesterone activates c-Src that is required for FAK activation and endothelial migration. As alternative mechanisms, other studies also indicate the central role of c-Src in endothelial migration by recruiting and activating downstream effectors like protein kinase C or matrix metalloproteinase [48,49].
This study indentifies that PI3K, a well-known downstream of c-Src [25], is activated by c-Src and is required for FAK activation. This is consistent with previous reports showing that the c-Src/PI3K/Akt pathway is implicated in Tyr 397 FAK phosphorylation in cancer cells [18,50]. The activation of PI3K/Akt results in the recruitment of the RhoA/ROCK-2, which is implicated in the regulation of endothelial functions such as cytoskeleton organization, motility, proliferation, adhesion and apoptosis [51]. Our findings do show that the activation of RhoA/ROCK-2 results in endothelial FAK phosphorylation and migration. In line with this, RhoA/ROCK cascade is shown to activate FAK in cardiac myocytes [27]. However, the mechanism through which RhoA/ROCK-2 activates FAK is still not completely understood. In this regard, it has been suggested that phosphorylated caveolin-1 relays signals from RhoA/ROCK-2 to FAK [52]. Caveolin-1 is regarded as a molecular switch for some of the extra-nuclear actions of sex steroids in vascular endothelial cells [53]. It would thus be interesting to interrogate the role of caveolin-1 in progesterone-induced FAK phosphorylation.
Of note, although it is shown that RhoA/ROCK cascade is the intermediate between PI3K to FAK, it is still possible that PI3K directly interacts with FAK and results in FAK activation, because the interaction of the p85 regulatory subunit of PI3K with FAK has been shown to trigger FAK phosphorylation [50,54]. Intriguingly, active FAK is able to interact with p85 to increase its activity, thus creating a positive feedback loop [55]. Therefore, further exploration on the relevance of endothelial PI3K and FAK is needed.
In this work, we observed the endothelial actions of natural progesterone, but not of synthetic progesterone such as MPA, the compound widely used in combined HRT. It has been documented that natural progesterone or synthetic progestins have a variable influence on endothelial function. For example, natural progesterone increases endothelial nitric oxide production whereas MPA is devoid of such action [56]. In non-human primates MPA has been shown to interfere with the atheroprotective effects of oestrogens, which does not happen with natural progesterone [57,58]. The intracellular signalling pathways recruited by progesterone and MPA are also divergent, even though they exert the similar biological action. In this regard, we have demonstrated that progesterone induces rapid activation of actin-binding protein moesin and endothelial or breast cancer cell migration through G protein, whereas MPA requires PI3K/Akt activation to complete the same effects [14,28]. Therefore, the effect on FAK activation and the signalling recruited by progesterone cannot be extended to other synthetic progestins. Future research will be necessary to address the regulatory effect of MPA on endothelial FAK activation.
In conclusion, it is found that progesterone promotes endothelial cell movement by facilitating the formation of FA complexes via the rapid activation of FAK, which is mediated by c-Src/PI3K/Akt and RhoA/ROCK-2 cascade. These findings provide new information on the biological actions of progesterone on human endothelial cells that are relevant for vascular function.