Regulatory Effect Of Caveolin-1 On Endothelial Cell Proliferation

Angiogenesis, the development of new blood vessels from preexisting capillary beds, is implicated in many physiological and pathological processes such as embryonic development, wound healing, and tumorigenesis. Endothelial cell proliferation is prerequisite for angiogenesis, and inhibition of endothelial mitogenic signaling is thus a principle target for anti - angiogenic therapy.The proliferation of endothelial cells is regulated by a number of angiogenic activators, inhibitors, and extracellular matrix components. Vascular endothelial growth factor ( VEGF) is a specific and potent mitogen for endothelial cells. VEGF is expressed in many kinds of tumor cells as well as normal tissue. In tumor cells, VEGF promotes angiogenesis and plays a role in tumor development. VEGF binds at least two receptor tyrosine kinases, of which VEGFR - 2 (KDR) seems to be the principle mediator of mitogenic signaling. Upon ligand binding, KDR is coupled to a number of signal transduction pathways, including the Ras - p42/44 MAP kinase pathway which stimulates endothelial cell proliferation, migration, and differentiation.Caveolin - 1, a 22KD integral membrane protein, is the marker protein of caveolae. There are 3 members in caveolin family: Caveolin - 1, caveolin -2, and caveolin - 3. Caveolin - 1 and caveolin - 2 associate to form hetero - oli-gomers, and are expressed abundantly in endothelial cells, adipocytes, epithelial cells, fibroblasts, where they presumably serve important roles.Recent studies have implicated a role for caveolin - 1 in angiogenesis. VEGF stimulation has been shown to down regulate caveolin - 1 and pretreat-ment with angiogenesis inhibitors blocks this effect. Moreover, over - expression of caveolin -1 has been shown to block VEGF dependent expression of Elk -1.Beliveau and colleagues have demonstrated that KDR cofractionates with caveo-lin -1 and that basal KDR and Src kinase activity is inhibited through direct interaction with caveolin - 1. However, the exact effect of caveolin - 1 on endo-thelial cells proliferation is not clear. Here we study the effect of up regulating caveolin -1 levels on endothelial cells proliferation and the cell cycle distribution as well as growth factor signaling and integrin - FAK signaling.Materials and methods1?. Materials and reagentsCaveoiin - 1, phospho - P44/42, GAPDH antibodies were purchased from BD Bioscience; P27kipl and Rb antibodies were purchased from Oncogene. H -Thymidine was obtained from Amersham BioSci. Gelatin was obtained from Sigma. Recombinant human vascular endothelial growth factor (VEGF165) was purchased from R&D Systems. 30% (w/v) Acrylamide and Scintillation liquid were purchased from National Diagnostics. TEMED was purchased from Fisher Scientific.2. Adenovirus infection of HUVEC cellsFor adenoviru& infection, cells were seeded in a 6 well plate coated with 0. 2% gelatin, incubated for 24 hours, washed with MCDB, then incubated with an appropriate titer of virus. After one hour, 2 ml complete medium was added to the cells. Various Ad - cav -1 and Ad - tTA Pfu were employed to achieve optimal levels of caveolin - 1 expression. One day post - infection, the cells were processed for immunofluorescence microscopy. As a control, Ad - GFP was used to co - infect HUVEC cells together with Ad - tTA.3. HUVEC cells proliferation assayAfter adenovirus infection, the HUVECs were incubated for 8 hours, washed in PBS, harvested, and collected by centrifugation at lOOOrpm for 5 minutes. The HUVECs were then resuspended in complete medium and seeded in a 96 well plate. After 24 hours, the cells were washed and starved with 0.5 % FBS MCDB overnight. The following day, the cells were stimulated with 10 ng/ ml VEGF or complete medium for 48 hours at 371!, 2 jiCi H3 -Thymidine/mlwas added to the cells upon the last 4 hours of incubation. The cells were then washed, and fixed in methanol and 10% TCA. The cells were then washed 3 times with H2O, and 50 jxl 0. IN NaOH was added to lyse the cells. Scintillation solution was added to the lysate and mixed, and the liquid scintillation was then counted.4. Effect of caveolin -1 on KDR phosphorylationAfter overnight starvation of the cells in 0. 5% FBS, the cells were stimulated with 10 ng / ml VEGF for 10 minutes, washed once in PBS, and lysed in boiling SDS sample buffer. The lysate was then boiled for 5 minutes. The samples were separated by SDS - PAGE gel electrophoresis and the proteins were transferred to membrane and immunobloted with phospho - KDR antibody.5. P42/44MAPK phosphorylation assayAfter infection, HUVECs were incubated for 36 hours, starved with 0.5% FBS 12 hours, and then stimulated with lOng/ml VEGF for 10 minutes. The cells were then lysed in boiling SDS - sample buffer and boiled for 5 minutes. The samples were resolved on a 12% SDS -PAGE gel, transferred to membrane and immunobloted with P42/44MAPK phosphorylation antibody.6. Careolin -1 co - immunoprecipitates with FAKHUVECs were washed 2 times with PBS, and lysed in RIPA buffer on ice for 20 minutes. Lysates were clarified by centrifuge at 5000rpm for 5mintes at 4°C. Protein extracts were incubated with 3 mg FAK antibody for 6 hours at 4t, then 40 |jlL of protein A/G agarose beads were added to each sample. After washing the beads with wash buffer, loading buffer was added to the beads and then boiled for 5 minutes. The co - immunoprecipitation complex was separated by SDS - PAGE gel electrophoresis and transferred to membrane and then blotted with caveolin -1 antibody.7. FAK phosphorylation assayHUVECs were seeded in a fibronectin coated 6 well plate and incubated for 30 minutes, after which the cells were lysed in boiling SDS sample buffer. The protein lysates were separated by 10% SDS - PAGE gel electrophoresis and transferred to membrane. The membrane was blotted with phospho - FAK and FAK antibody.8. p27kipl protein level assayUpon reaching 60% confluence, the cells were infected with adenovirus. 12 hours after infection, the cells were starved in 0. 5% FBS overnight, then stimulated with lOng/ml VEGF for 0, 8, 12, and 24 hours. The cells were washed with PBS once, and then lysed in boiling SDS - sample buffer and boiled for 5 minutes. The samples were resolved on a 12% SDS - PAGE gel, transferred to membrane and immunobloted with p27kipl antibody.9. HUVEC cell cycle analysisUpon reaching 60% confluence, the cells were infected with adenovirus. 8 hours post infection, the cells were washed in PBS, starved in 1 % FBS MCDB for 18 hours, then stimulated with growth medium for 16 hours. At these two time points, the cells were collected and the cell cycle distribution was analyzed.Results1. Over - expression of caveolin -1 by infection of HUVEC cells with adenovirusIn order to investigate the effect of caveolin - 1 on endothelial cell proliferation, we used adenovirus as a vector to express exogenous caveolin - 1. We found that infection with 200 pfu Ad - cav - 1 and 200 pfu Ad - tTA, nearly 90% of the cells express the exogenous caveolin - 1, and when the cells were infected with 20 pfu Ad - GFP and 100 pfu Ad - tTA, more than 90% of the cells express GFP protein. This system was subsequently used to study the effect of caveolin - 1 on endothelial cells proliferation.2. Over - expression of caveolin -1 dramatically inhibits the proliferation of HUVEC cellsFirst, to investigate the effect of caveolin -1 over - expression on HUVEC cells proliferation, we examined the incorporation of H3 - Thymidine as an index of de novo DNA synthesis. Briefly, HUVECs in a six well plate were infected with adenovirus that express GFP or caveolin -1, and then seeded in a 96 well plate, starved with 0. 5% FBS overnight, then stimulated with VEGF or com-plete medium (with 15%FBS). Stimulation of HUVECs with lOng/ml VEGF resulted in a nearly 2 folds increase in thymidine incorporation of control cells o-ver base line levels. Thymidine incorporation was blunted in VEGF stimulated cells over - expressing caveolin - 1, with a rate of incorporation nearly two - fold less than GFP - expressing or mock treated cells. Similarly, the rate of H -thymidine incorporation upon serum stimulation was three - fold less in caveolin - 1 over - expressing cells compared to control cells. These results suggest that VEGF - and serum - induced proliferation rates are inhibited by caveolin -lover - expression.3. Over - expression of caveolin -1 inhibits the activity of VEGF receptor KDRlike other growth factor receptors bound with ligand, KDR forms a dimer and is autophosphorylated on multiple tyrosine residues. These sites can be involved in the regulation of kinase activity or serve as binding sites for SH2 - domain - containing signaling proteins. Phosphorylation of tyrosine 1054 in the activation loop is required for activation of KDR and its intrinsic tyrosine kinase activity. We used an antibody that specifically recognizes tyrosine 1054 phosphorylation as an index of KDR activity. After starvation, very little phosphorylation of KDR is detectable by Western blot in cells whether over - expressing caveolin - 1 or not. After stimulation, however, control cells showed a robust phosphorylation of KDR whereas caveolin - 1 over - expression abrogated this effect.4. Over - expression of caveolin -1 inhibits the activity of p42/44 MAP kinaseCaveolin - 1 over - expression has been shown to inhibit mitogenic signaling and proliferation in a number of cell lines. To determine whether or not a similar effect is seen in endothelial cells, we over - expressed caveolin - 1 in HUVECs. The effect of caveolin - 1 over - expression on VEGF - induced signaling was determined by measuring the degree of phosphorylation of p42/44 MAP kinase by Western blot. Unstimulated cells showed negligible p42/44 MAPK phosphorylation, while VEGF stimulation of mock - treated and GFP -expressing HUVECs resulted in a swift and robust phosphorylation of p42/44MAP kinase. In contrast, cells over - expressing caveolin - 1 showed a diminished response to VEGF stimulation, with decreased phosphorylation of an overall pool of p42/44 MAPK equal to that of mock - treated or GFP - expressing HUVECs.5. Caveolin - 1 over - expression inhibits fibronectin induced FAK phosphorylationTo determine whether caveolin - 1 affects the integrin - FAK pathway, we explored whether caveolin -1 interacts with FAK by co - immunoprecipitation. Indeed, we found that caveolin -1 co - immunoprecipitates with FAK. We then studied the effects of caveolin - 1 over - expression on FAK tyrosine 397 phosphorylation. Compared with mock and GFP expressing cells, the caveolin -lover - expressing cells demonstrated a significant blunting of FAK phosphorylation upon fibronectin stimulation.6. Over - expression of caveolin -1 up - regulates the cyclin - dependent kinase inhibitor p27 KiplWe next analyzed the effect of caveolin - 1 over - expression on the expression of p27Kipl, an important cell cycle regulatory protein that prevents the Gl/ S transition in mammalian cells. MAPK has been demonstrated for phosphorylate p27Kipl indirectly, which might trigger degradation of p27kipl through the ubiquitin pathway. Western blot shows that p27Kipl levels are higher in caveolin - 1 over - expressing cells compared to control levels 8 hours after stimulation by VEGF. By 12 hours after stimulation control cells , p27Kipl levels are nearly equal to caveolin - 1 over - expressing cells, suggesting a transient decrease in p27Kipl levels due to VEGF stimulation.7. Over - expression of caveolin - 1 arrests endothelial cells in the G0/G1 phase of the cell cycleGiven the inhibition of MAP kinase activity and up regulation of p27kipl level, we hypothesized that cavelin - 1 over - expression inhibits cell proliferation by inducing GG/G1 arrest in HUVECs. To test this, HUVECs were infected with caveolin -1 or GFP adenoviruses, or mock infected and serum starved to arrest the cell cycle at the G0/G1 phase. Upon addition of serum, GFP or mock infected cells entered the S phase at a rate nearly twofold greater than that of thecaveolin -1 over - expressing cells (35% vs. 19. 6% ). Compared with control cells with or without GFP expression, the caveolin - 1 over - expression resulted in an appreciable arrest at G0/G1 phase (70% vs. 52% ). Caveolin - 1 over -expression was shown to inhibit VEGF and serum induced proliferation. This result suggests that caveolin - 1 over - expression in part through inactivation of MAP kinase inhibits endothelial cell cycle progression.DiscussionThe results obtained in the present study demonstrate for the first time that over - expression of caveolin - 1 protein inhibits HUVECs proliferation through inhibition of the cell cycle progress. This result is constant with evidence of endothelial cells hyperplasia in caveolin - 1 null mice. The mechanism behind this is unclear, so we studied the molecular events associated with cavolin - 1 over -expression in endothelial cells.1. Caveolin -1 inhibits the Ras -p42/44 MAPK signal transduction pathwayFirst, over - expression of caveolin - 1 inhibits VEGF - induced KDR phosphorylation at tyrosine residue 1054. VEGF is one of the most potent mito-gens of endothelial cells. There are at least two VEGF receptors: KDR and Fit -1. KDR plays an important role in endothelial proliferation and can form im-munoprecipitation complexes in BAEC cells. After the activation of KDR by VEGF binding, the signal is transduced to downstream proteins, whereupon p42/44 - MAPK is activated. We found that caveolin -1 inhibits the activity of p42/44MAPK. In agreement with this, p42/44MAPK has been shown to be hy-peractivated in fibroblast cells in caveolin -1 null mouse compared to wild type mice.2. Caveolin -1 inhibits the integrin - FAK signal transduction pathwayAs a scaffolding protein, caveolin - 1 recognizes and binds signal molecules and always inhibits their activity. Our co - immunoprecipitation experiment demonstrated that caveolin -1 and FAK can form immunoprecipitation complexes. After checking the FAK amino acid sequence, we found caveolin - 1 bind-ing sequence in FAK: 251YRFDKECFKC261. This result suggests that caveolin -1 and FAK can interact directly. Tyrosine 397 is the autophosphorylation site of FAK and is involved in its initial activation. Over - expression of cavolin -1 inhibits the phosphorylation of tyrosine 397, further evidence that caveolin -1 and FAK may interact directly. After phosphorylation of tyrosine 397, FAK interacts with Src and is phosphorylated by Src at other sites, then interact with Grb2, and subsequently activates the Ras - MAPK pathway. Other research shows that cavolin -1 interacts with Src and inhibits its phosphorylation. Thus caveolin -1 inhibits the FAK signal pathway through inhibition of the activity of FAK and Src.3. Cavolin -1 affects the level of CDK inhibitor p27kiplRecent studies have shown that the change of p27kipl levels was due to phosphorylation of p27kipl, which is subsequently degraded via the ubiquitin pathway. Our experiment shows that p27kipl was up - regulated when caveolin -1 is over - expressed in endothelial cells. One possible mechanism is that caveolin - 1 inhibits p27kipl phosphorylation by inhibiting the Ras - MAPK signaling pathway.4. The significance of cavoelin - 1 inhibition of endothelial cell proliferationAngiogenesis plays a critical role in tumor growth and metastasis. In order to grow over a few cubic millimeters in size, the tumor needs blood vessels to supply nutrition and oxygen. Tumor cells secrete some angiogenesis activators, such as VEGF, bFGF, and these induce the endothelial cells in nearby vascular beds to proliferate. On the other hand, endothelial cells in normal tissue remain quiescent. A large body of evidence has demonstrated that suppression of angiogenesis effectively blocks the tumor growth and metastasis. Endothelial cell proliferation is a prerequisite step in angiogenesis, so inhibition of angiogenesis through inhibition of endothelial cells proliferation is an attractive therapeutic goal.ConclusionCaveolin - 1 inhibits the Ras - p42/44MAPK pathway and integrin - FAK pathway, up regulates p27kipl level in endothelial cells, ultimately inhibits endothelial cell proliferation through arrest the cell cycle at GO/Gl phase.