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Reactive oxygen species (ROS) contribute to a number of cardiovascular disease pathologies including hypertension, atherosclerosis, and restenosis (1). When blood flow through an artery is impeded by a blockage, treatment frequently includes balloon angioplasty and the insertion of a wire stent. This procedure often causes injury to the artery wall, which in turn induces vascular smooth muscle cell (VSMC) migration within the artery wall and the formation of a neointima in an effort to heal the wound. VSMC migration is also important in atherosclerotic lesion formation. The signaling pathway that mediates this process and the proteins involved in this pathway are the focus of this study.
Several proteins are known to play a role in focal adhesion assembly and disassembly, one of which is a protein tyrosine phosphatase (PTP) known as PTP-PEST (5). PTP-PEST is strongly associated with focal adhesion turnover (3). We hypothesize that PTP-PEST activity and its role in focal adhesion disassembly are regulated by ROS in VSMCs.
Other proteins that affect focal adhesion turnover include Src, focal adhesion kinase (FAK), Pyk2, p130Cas, and RhoA. Modification of these proteins via phosphorylation or their associations has been shown to regulate ECM-actin linkages by altering their affinities for integrin proteins of focal adhesions.
One source of ROS production in VSMCs are the NADPH oxidase enzymes known as Nox1 and Nox4. The recent discovery of a possible Nox4 regulatory subunit, termed NoxR1 for Nox regulator 1, by the Griendling lab provides new evidence that Nox4 may utilize unique cytosolic regulatory subunits for its activity. Overexpression of NoxR1 increases Nox4-dependent oxidase activity, superoxide production, and hydrogen peroxide production (unpublished data).
Preliminary results from the Griendling lab also demonstrate that the knockdown of NoxR1 using small interference RNA (siNoxR1) and the overexpression of NoxR1 causes striking phenotypic changes in VSMCs, similar to the phenotypes associated with PTP-PEST overexpression and knockdown. Their results suggest that when NoxR1 activates Nox4, it regulates focal adhesion maturation. Furthermore, overexpression of NoxR1 not only increases ROS production, but results in increased oxidation of PTP-PEST at its reactive cysteine residues. VSMCs overexpressing NoxR1 also show significantly increased levels of active RhoA, increased focal adhesion number and size, increased stress fiber formation, and impaired VSMC migration. In contrast, VSMCs treated with siNoxR1 have decreased focal adhesions, decreased stress fiber formation, and decreased VSMC migration. All of these preliminary results together have led us to hypothesize that overexpression of NoxR1, through increased ROS results in an increase in Src and FAK association, as well as increased PTP-PEST oxidation, both of which result in increased focal adhesion and stress fiber formation, a decrease in focal adhesion turnover, and decreased VSMC migration.
Hypothesis
We hypothesize that ROS production via Nox4/NoxR1 regulates focal adhesion turnover by influencing the association between Src and FAK.
Cell Culture – To determine whether overexpression of NoxR1 alters Src and FAK phosphorylation, VSMCs from male Sprague-Dawley rat thoracic aortas were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% calf serum, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells passages 8 to 12 were used for experiments. VSMCs were grown to 70% confluence and were treated with no adenovirus (0 Ad) or transduced with adenovirus expressing Myc tagged NoxR1 (AdNoxR1) or control adenovirus expressing GFP (AdCMV) for 72 hours in serum free DMEM. Cells were then lysed and lysates were resolved on SDS-PAGE.
Immunoprecipitation – VSMCs were grown to ~70% confluence and were made quiescent by serum-deprivation for 72 hours. Cells were lysed in Hunter's buffer and protein was determined for the whole cell lysates using a Bradford assay. Total cell lysates were incubated with equal amounts of either rabbit anti-Src (Cell Signaling) or an equal amount of rabbit IgG antibody and subsequently with protein A/G Plus agarose beads (Santa Cruz). Beads were washed five times with standard lysis buffer, analyzed by SDS-PAGE, and immunoblotted with anti-FAK (Cell Signaling) and anti-Src (Santa Cruz) antibodies.
siRNA transfection – VSMCs were plated on collagen-coated dishes at 50% confluence were either left untreated or were treated with small interfering RNA (siRNA) duplexes specific for rat NoxR1 (siNoxR1). Scrambled, non-targeting siRNAs (siControl) served a control to validate the specificity of siNoxR1. siRNAs were introduced into the cells by transfection using Oligofectamine. Cells were harvested after being maintained in OPTI-MEM media for 72 hours. An immunoprecipitation was then performed as described above.
Western blotting – was performed using antibodies for the following: phospho-Src antibody (Cell Signaling), total Src antibody (Cell Signaling), phospho-p130Cas (Cell Signaling), total p130Cas (Upstate), phospho-Pyk2 (Cell Signaling), total Pyk2 (Cell Signaling), anti-Myc-tag (Cell Signaling), and NoxR1 antibody (Griendling Lab). Band intensity was quantified by densitometry of immunoblots by using NIH Image J, version 1.36.
Src-FAK Association
Previous experimental findings show that the association between Src and FAK is essential for focal adhesion (FA) maintenance, a critical factor in cellular modulation of migration, growth, survival, and gene expression. For example, FAK null fibroblasts have larger, more stable FA and lose random migration, suggesting the essential role of FAK in FA turnover. Our overall objective is to investigate the mechanism by which NoxR1 influences FA turnover. To test this we first investigated how NoxR1 overexpression affected the phosphorylation states of Src and FAK. While there was no significant change in Src or FAK phosphorylation due to NoxR1 overexpression, preliminary immunoprecipitation studies indicate that there is a basal association between Src and FAK and that this association is ROS sensitive, as it is diminished in VSMCs treated with diphenyleneiodonium (DPI). Our current focus is to investigate how knockdown of NoxR1 affects this association. This is tested by performing a co-immunoprecipitation in VSMCs treated with siRNA against NoxR1 (siNoxR1).
Overexpression of NoxR1 results in the increased oxidation of the protein
tyrosine phosphatase, PTP-PEST.
Overexpression of NoxR1 leads to no significant changes in the
phosphorylation of Src or FAK.
Src and FAK associate at a basal state in quiescent VSMCs.
Knockdown of NoxR1 with siRNA induces a decrease in the association
between Src and FAK.
It had been demonstrated in other systems that when PTP-PEST is inactivated, targets such as Src, p130Cas, Pyk2, and FAK are hyper-phosphorylated. Recent preliminary data show that when NoxR1 is overexpressed in VSMCs, PTP-PEST is oxidized. Our initial hypothesis was that oxidation of PTP-PEST would result in decreased phosphatase activity and hyper-phosphorylation of these downstream targets. However, our recent data indicate that Src, p130Cas, and Pyk2 are not likely downstream of oxidized PTP-PEST and upstream of active RhoA, as NoxR1 overexpression did not result in an increase in phosphorylation of any of these targets.
There are several possible explanations for the lack of hyper-phosphorylation of these targets in response to NoxR1 overexpression. Although experiments have demonstrated that PTP-PEST is oxidized when NoxR1 is overexpressed, we have yet to confirm that there is a subsequent decrease in PTP-PEST activity. This will be a focus of future experiments, since it is possible that PTP-PEST oxidation may cause an increase in phosphatase activity in this system. An alternative explanation is that PTP-PEST normally dephosphorylates and inactivates another phosphatase downstream. If PTP-PEST is oxidized, according to our model, this hypothetical phosphatase would not be inhibited by PTP-PEST, and p130Cas, Src, and Pyk2 would be dephosphorylated.
Because the downstream targets are not hyper-phosphorylated in response to NoxR1 overexpression, we chose to explore other possible mechanisms of NoxR1/Nox4 regulation of cell migration. FA turnover and subsequent cell migration has been shown to be affected by the increased association of Src and FAK; therefore, it is the new focus of this study.
Our current experimental results demonstrate that NoxR1 regulation of Nox4, and subsequent ROS production, is upstream of Src, FAK, and PTP-PEST. We have shown that NoxR1 may potentially mediate its effects on focal adhesion turnover through the regulation of Src and FAK protein association. We demonstrated that Src and FAK associate in quiescent VSMCs and that knockdown of NoxR1 using siRNA resulted in a decrease in the association between Src and FAK, suggesting that NoxR1/Nox4-dependent production of ROS affects VSMC migration through regulation of this critically important protein:protein association. Our data also suggest that some of the effects on focal adhesion turnover and VSMC migration may be mediated by simultaneous oxidation of the protein tyrosine phosphatase, PTP-PEST, which inactivates the phosphatase. These data confirm that NoxR1 and ROS are critical regulatory components in the process of VSMC migration.
Repeat the co-immunoprecipitation of Src and FAK in siNoxR1
treated VSMCs.
Investigate the effects of NoxR1 overexpression on the
association between Src and FAK in VSMCs.
Determine if there is a direct link between Src-FAK association
and VSMC migration.
I would like to thank Dr. Kathy Griendling and Alicia Lyle for laboratory assistance, data analysis advice, and mentorship. This material is based upon work supported by the Howard Hughes Medical Institute Grant No. 52005873 and by the National Institutes of Health Grant No. HL38206.
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