The nonreceptor tyrosine kinase, Pyk2, is an important regulator of EC barrier function and angiogenesis (23
). Recently, it has also been implicated in proinflammatory gene expression and leukocyte TEM (26
); however, the mechanistic basis of these responses is only partially understood. Because NF-κB is an essential regulator of genes involved in TEM of leukocytes, the present study was undertaken to address the role of Pyk2 in the mechanism of NF-κB activation in ECs. Our data show that Pyk2 is an important determinant of thrombin-induced EC inflammation, and that it mediates this response by activating IKK to promote the release and the transcriptional capacity of RelA/p65, and additionally by facilitating the nuclear translocation of released RelA/p65. Importantly, Pyk2 also controls NF-κB activation induced by TNF-α, suggesting that Pyk2 is a general regulator of NF-κB signaling in the endothelium.
We began our investigation by defining the time course of Pyk2 activation by thrombin. We noted a biphasic phosphorylation of Pyk2: an early and transient phase occurring between 1 and 5 minutes, followed by a late phase that begins at 30 minutes and is sustained for up to 2 hours after thrombin stimulation. The early phase of Pyk2 activation is in accord with a previous report (21
) showing a similar time course of Pyk2 phosphorylation; however, the late phase was not detected in the earlier study, as Pyk2 phosphorylation was followed only up to 10 minutes after thrombin challenge. Thus, our data reveal a second phase (in addition to the early phase) of Pyk2 phosphorylation, and are consistent with two-stage mobilization of Ca2+
, a requirement for Pyk2 activation, induced by thrombin in ECs (37
). Pyk2 activation was also observed in LPS-stimulated human umbilical vein ECs and human dermal microvascular ECs (26
), as well as in cyclic strain-exposed bovine aortic ECs (22
). Thus, Pyk2 appears to be responsive to the proinflammatory environment in ECs from different vascular beds.
The activation of Pyk2 led us to assess its involvement in NF-κB activation. To this end, we evaluated the effect of RNAi knockdown of Pyk2 on NF-κB–dependent reporter activity. Depletion of Pyk2 abolished thrombin-induced NF-κB activity, indicating the requirement of Pyk2 in the response. Our findings are consistent with those of earlier studies indicating the involvement of Pyk2 in G protein–coupled receptor (GPCR)-mediated signaling and NF-κB activation (18
). We also examined if the impaired NF-κB activity secondary to Pyk2 knockdown leads to reduced expression of VCAM-1 and MCP-1. A significant decrease in the expression of these genes was observed after thrombin stimulation of Pyk2-depleted cells. Notably, the role of Pyk2 in NF-κB–dependent gene expression is not restricted to thrombin and the endothelium, but can also be induced by a variety of stimuli and in different cell types. Consistent with this, we found that depletion of Pyk2 was effective in inhibiting TNF-α–induced NF-κB activity in ECs. Other studies showing a role of Pyk2 in LPS-induced expression of IL-8 and MCP-1 in ECs, carbacol-induced NF-κB–dependent reporter activity in HeLa cells, and LPS- and peptidoglycan-induced IL-1β expression in macrophages (26
) further underscore the importance of Pyk2 as a common mediator of NF-κB signaling.
Our study yields the mechanistic basis of NF-κB activation by Pyk2. We demonstrate that Pyk2 mediates NF-κB activation by promoting the release, increasing the transcriptional capacity, and facilitating the nuclear translocation and DNA binding of RelA/p65. Nuclear translocation and DNA binding of RelA/p65 is contingent upon its release secondary to Ser32 and Ser36 phosphorylation, and, subsequently, degradation of IκBα. Consistent with this, Pyk2 knockdown impaired the phosphorylation of IKK and IκBα. Similarly, depletion of IKKα or IKKβ also inhibited IκBα phosphorylation and degradation. Intriguingly, however, Pyk2 depletion had no effect on IκBα degradation by thrombin. This does not seem to be the result of a nonspecific effect of Pyk2-siRNA, as IκBα level remained intact in unstimulated cells (). One possible explanation could be that a Ser32 and Ser36 phosphorylation–independent (i.e., IKK independent) mechanism of IκBα degradation is activated by thrombin in the absence of Pyk2. Indeed, studies have shown that phosphorylation of IκBα-PEST (proline–glutamic acid–serine-threonine) sequences by CKII-dependent mechanism also leads to its degradation (41
). It is likely that the inhibitory effect of Pyk2 depletion on IKK-dependent IκBα degradation is masked by CKII-dependent degradation of IκBα that may be activated by thrombin in the absence of Pyk2. Such a possibility is further supported by the requirement of Ca2+
for the latter mode of IκBα degradation (41
). Because Ca2+
is also required for Pyk2 activity, it is plausible that the depletion of Pyk2 increases the availability of Ca2+
to facilitate CKII-mediated PEST sequence phosphorylation–dependent degradation of IκBα by thrombin. In support of this model, we found that RNAi knockdown of CKII was effective in protecting IκBα degradation only in cells depleted of Pyk2 (Figure E4). However, additional studies are required to unequivocally address this possibility.
Interestingly, Pyk2 knockdown was associated with impaired nuclear translocation and DNA binding of RelA/p65 in the face of IκBα degradation. These results suggest that Pyk2 may also be involved in facilitating the translocation of released RelA/p65 to the nucleus. We recently found that dynamic changes in actin cytoskeleton induced by thrombin via RhoA/Rho-associated kinase (ROCK)/Cofilin pathway are necessary for nuclear translocation of RelA/p65 (43
). Given the involvement of Pyk2 and its downstream kinase c-Src in activating RhoA/ROCK pathway (19
), it is possible that Pyk2 promotes nuclear translocation and, consequently, DNA binding of RelA/p65 by causing alterations in the actin cytoskeleton. Consistent with this possibility, inhibition of Pyk2 by tyrphostin A9 attenuates thrombin-induced Ser3 phosphorylation and, thereby, inactivation of cofilin1 (K.M.B., unpublished results), an actin-binding protein that occupies a central position in Rho–actin pathway mediating RelA/p65 nuclear translocation (43
). However, in view of the reports that Pyk2 can also function downstream of RhoA/ROCK (47
), the other possibility that RhoA/ROCK engages Pyk2 to mediate the above responses cannot be excluded. Further studies are needed to establish how Pyk2 and RhoA/ROCK are linked in the mechanism of NF-κB activation by thrombin.
An important event implicated in enhancing the transactivation potential of RelA/p65 includes its phosphorylation on serine residues, including Ser536 (10
). We previously showed that thrombin induces Ser536 phosphorylation of RelA/p65, and that this response is mediated, in part, by p38 MAPK (34
). Because Pyk2 is required for p38 MAPK activation (50
), and Pyk2 depletion reduces Ser536 phosphorylation of RelA/p65, we reasoned that Pyk2 may act upstream of p38 MAPK to mediate this response (RelA/p65 phosphorylation). However, our data that thrombin activates p38 MAPK in a Pyk2-independent manner argues against the involvement of Pyk2/p38 MAPK in RelA/p65 phosphorylation, and suggests the existence of an additional pathway that Pyk2 uses for this response. Indeed, we found that Pyk2 engages IKK (IKKα and IKKβ) to promote RelA/p65 phosphorylation and, thereby, NF-κB signaling induced by thrombin. Our findings that inhibition of p38 MAPK fails to prevent thrombin-induced IκBα phosphorylation at Ser32 and Ser36 (Figure E2B), an IKK-mediated event required for IκBα degradation and subsequently nuclear DNA binding of RelA/p65 (10
), also argues against the engagement of p38 MAPK upstream of IKK to mediate RelA/p65 phosphorylation. Thus, the inability of Pyk2 to mediate thrombin-induced p38 MAPK activation compared with an important role of Pyk2/p38 MAPK signaling in LPS-induced IL-8 and MCP-1 expression and vascular endothelial growth factor–induced EC migration (26
) reveals a stimulus-specific activation of p38 MAPK by Pyk2 in ECs, and shows a fundamental signaling difference between thrombin and other receptors (GPCR versus LPS and vascular endothelial growth factor). It should also be stressed that the dominant inhibitory effect of Pyk2 depletion on thrombin-induced NF-κB activation and EC inflammation may derive from the ability of Pyk2 to regulate RelA/p65 at multiple levels (release of RelA/p65 from IκBα, nuclear translocation, and the transcriptional capacity of the released RelA/p65). Unlike Pyk2, p38 MAPK contributes to NF-κB activity via RelA/p65 phosphorylation alone. Moreover, in a separate ongoing study, we have found that Ser536 phosphorylation is dependent upon IκBα degradation (K.M.B., unpublished results). Because p38 MAPK is not required for IκBα degradation (10
), its contribution to RelA/p65 phosphorylation and, thereby, EC inflammation may rely on Pyk2-dependent IκBα degradation induced by thrombin.
In summary, this study identifies Pyk2 as a critical determinant of NF-κB activation and EC inflammation. We show that Pyk2 engages IKK to liberate RelA/p65 from IκBα and empower the liberated RelA/p65 with the transcriptional capacity. Additionally, Pyk2 is engaged to facilitate the nuclear accumulation and DNA binding of the released RelA/p65. Together, these events serve to promote EC inflammation. Thus, specific targeting of Pyk2 may be an effective anti-inflammatory strategy in vascular diseases associated with EC inflammation and intravascular coagulation.