Cross Talk Among Smad, MAPK, and Integrin Signaling Pathways Enhances Adventitial Fibroblast Functions Activated by Transforming Growth Factor–β1 and Inhibited by Gax
Objective— We investigated whether Smad, mitogen-activated protein kinase (MAPK), and integrin signaling pathways cross-talk to enhance adventitial fibroblast (AF) bioactivity, which was activated by transforming growth factor (TGF)-β1 and inhibited by Gax.
Methods and Results— Cultured AFs were stimulated with Ad-Gax, TGF-β1, and siRNA-Gax. Assays for AFs viabilities demonstrated that TGF-β1 and siRNA-Gax enhanced AFs proliferative, migratory, and adherent abilities, whereas Gax counteracted TGF-β1–activated actions. Flow cytometry revealed that TGF-β1 and siRNA-Gax increased S phase cells; however, Gax decreased AFs in the S phase and increased those in the G0-G1 and apoptotic phases. RT-PCR, Western blotting, and immunocytochemistry showed that TGF-β1 and siRNA-Gax upregulated the expression of cytokines in Smad, MAPK, and integrin signaling pathways, and downregulated that of p15, p16, and p21. Conversely, Gax induced downregulation of these cytokines and upregulation of p15, p16, and p21. Thus, these signaling pathways cross-talk to enhance AF bioactivity; Gax effectively counteracts TGF-β1 effects, blocks the cross-talk of these pathways, inhibits AF functions, and increases AF apoptosis.
Conclusions— Our findings indicate that cross-talk among Smad, MAPK, and integrin signaling pathways may account mainly for the mechanism of AF functions. Gax is a promising therapeutic gene for dissecting the signaling pathways controlling AF bioactivities.
Previously, vascular intima and media were emphasized in research into vascular proliferation such as vascular restenosis and atherosclerosis. Recent studies, however, have revealed that vascular intimal proliferation is not only caused by the intima injury but also produced by the intervention in vascular adventitia. AF, as the major cell component in vascular adventitia, can respond to diverse stimulation and result in vascular stenosis by proliferation, migration, phenotype transformation, adhesion, cytokine excretion, and epimatrix formation after activation.1,2
Transforming growth factor (TGF)-β1 is an important pleiotropic cytokine with powerful effects on fibroblast bioactivity and is closely associated with vascular adventitial remodeling.3,4 TGF-β1 can accelerate AF proliferation and induce transdifferentiation of AF into myofibroblast, which is involved in vascular restenotic lesions by modulating mesenchymal cells growth, augmenting extracellular matrix (ECM) proteins synthesis, and facilitating neointima formation. Conversely, anti–TGF-β1 inhibited AF proliferation and harmful vascular remodeling. TGF-β1 can activate the Smad pathway, as well as other signaling pathways such as MAPK and integrin pathways, and induce cross-talk between the Smad pathway and other signaling pathways in some kinds of cells.5–8 However, the mechanism of TGF-β1 regulating AF bioactivity remains unclear.
Vascular remodeling occurs during normal angiogenesis and in various pathological states. Homeobox-containing genes encode transcription factors, regulate cell growth, differentiation, and migration, and play an essential role in the basic processes of embryogenesis and development.9–11 Homeobox transcription factors are not only key regulators of cell viability but also the controllers of some cytokines expression, which is associated with abnormal proliferation of many types of cells. Growth arrest–specific homeobox (Gax) is a unique homeobox gene among nuclear transcription inhibitors, at its highest levels in quiescent cells, and downregulated after mitogen activation. Gax expression is largely confined to adult cardiovascular tissues, with remarkable effects on inhibiting proliferation of vascular endothelial cells (VECs) and smooth muscle cells (VSMCs).9–11 However, whether Gax is endogenously expressed in AF and regulates its proliferation is unclear. We aimed to examine whether TGF-β1 activates a signaling transduction network that enhances AF proliferation and whether Gax effectively blocks the cross-talk of these pathways and inhibits AF bioactivities.
Materials and Methods
Detailed material and methods are described in the supplemental data (available online at http://atvb.ahajournals.org).
Adenoviral Vectors and siRNA Preparation
The rat Gax plasmid and pAdeasy-1 system with GFP were kind gifts of Dr Kenneth Walsh (St. Elizabeth’s Medical Center, Boston, Mass)9 and Dr Bert Vogelstein (The Howard Hughes Medical Institute, Baltimore, Md).12 Gax was amplified by PCR13 with the primers 5′-GGAAGCTTTCATAAGTGTGCGTGCTCAG-3′ and 5′-AAGGTACCATGGAACACCCCCTCTTTGGC-3′. The recombinant Ad-Gax and Ad-GFP adenoviral vector was constructed by 2-step transformation.12 The siRNAs targeting the Gax (GenBank number: NM_017149.1) with duplexes (siRNA-Gax) 5′-AAGGUAGGACAUGUGGUCAGAUCUU-3′ and 5′-AAGAUCUGACCACAUGUCCUACCUU-3′ were chemically synthesized from Integrated DNA Technologies Inc.
Cell Culture and Transfection
Adventitia was carefully decorticated from New Zealand white rabbit thoracic aorta. Cells were cultured by a modified explant method.14 After the AF purity was identified, cultured cells were transfected with viral liquid10,11 or siRNA-Gax with lipofectamine 2000 (Invitrogen) following the manufacturer’s instruction, and these cells were incubated and observed at different time points according to different experimental purposes.
Experimental Cell Grouping
The cells were serum-starved and synchronized for 24 hours. To observe the effects of TGF-β1 and Gax on AF functions, AFs in the experiment of Gax overexpression were grouped into Ad-GFP, Ad-GFP+TGF-β1, Ad-Gax+TGF-β1, and Ad-Gax groups. In the experiment of Gax low expression, AFs were grouped into AF (untreated), TGF-β1, siControl (negative siRNA duplexes for Gax), siRNA-Gax, siControl+TGF-β1, and siRNA-Gax+TGF-β1 groups. In culture medium, the final concentration of TGF-β1, viral liquid, and the short RNA duplexes was 5 ng/mL, 300 MOI, and 50 pmol/mL, respectively.
After stimulation for 48 hours, total RNA was extracted from the cultured AFs with Trizol and MMLV. mRNA was detected by semiquantified RT-PCR.13,15 Primers and the conditions for RT-PCR were shown in the supplemental data. The cytokine expression intensity was represented by a relative coefficient, cytokine OD/GAPDH OD.
After treatment with transfectants, AFs were harvested and washed 3 times with ice-cold PBS, then were lysed in buffer containing protease inhibitors and the total protein was extracted and detected by Western blotting.14,16 The expression of cytokine protein was demonstrated by the ratio of integral optical density (IOD) between cytokine and β-actin.
Assays for Cell Vitality
After treatment for 48 hours, 5-bromodeoxyuridine (Brdu) incorporation was measured by the improved Kahler method.17 After transfection for 24 hours, MTT and adhesion assays were respectively performed as described previously.11,17 After intervention for 72 hours, migration measurement was assayed by Sarker scratch method.18,19
Cell cycle analysis was performed as described previously.11 At 48 hours postintervention, with use of a FACScan flow cytometry (FCM) analysis system, cell cycle and apoptosis analyses were performed.
Immunochemical staining was performed as described previously.14 Tetraethylrhodamine isothiocyanate (TRITC)-labeled AFs were viewed as positive cells. IOD was the OD of positive cells multiplying the positively stained area. The mean IOD of antibody-labeled cell staining to that of PBS control was compared.
Results are presented as mean±SD and analyzed with SPSS 11.5. Comparisons among groups involved the use of ANOVA. Ratio comparison involved chi-square analysis. P<0.05 was considered statistically significant. All experiments were repeated at least 3 times.
The resulting vector was confirmed as correctly recombined Ad-Gax (supplemental Figure I, available online at http://atvb.ahajournals.org), and the Gax sequence was confirmed by DNA sequence. The titer of Ad-Gax was adjusted to 500 MOI. Based on the effects of different titer of Ad-Gax on AF function (supplemental Figure II), the optimal concentration of Ad-Gax in experimental groups used was 300 MOI.
AF Identification, Infection, and Gax Protein Expression
All cultured cells demonstrated negative monoclonal antibody staining for α-actin and positive monoclonal antibody staining for vimentin, which suggested 100% purity of cultured AFs (Figure 1A and 1B). Under normal conditions, AFs poorly expressed Gax protein (Figure 1C and 1D). At 12 hours after infection, almost all AFs were infected with Ad-Gax (Figure 1E and 1F). At 24 hours after infection, Gax was overexpressed mainly in nuclei (Figure 1G through 1I). After Ad-Gax infection, Gax protein expression peaked at 24 hour and started to decrease slightly thereafter (Figure IIIA, available online at http://atvb.ahajournals.org). At 24 hours after siRNA, Gax expression was knocked down to less than 20% with 50 pmol/mL of siRNA-Gax (supplemental Figure IIIB).
Effects of TGF-β1, Gax and siRNA-Gax on AF Proliferation
AF proliferative activity was calculated by BrdU-labeled AF counting (Figure 2, supplemental Figure IVA) and MTT assay (supplemental Figure IVB and IVC). The proliferative ratio and OD were significantly higher in the Ad-GFP+TGF-β1 group than those in the Ad-GFP group (Figure 2, Figure IVB), higher in the TGF-β1 and siRNA-Gax groups than those in the AF and siControl groups, higher in the siRNA-Gax+TGF-β1 group than those in the TGF-β1 and siRNA-Gax groups and higher in the siRNA-Gax group than those in the TGF-β1 group (supplemental Figure IVA and IVC). The Ad-Gax+TGF-β1 and Ad-Gax groups had a lower proliferative ratio and OD than the Ad-GFP+TGF-β1 group. Thus, both TGF-β1 and siRNA-Gax independently and synergistically stimulated AF proliferation and Gax knockdown had a more effective action than TGF-β1 stimulation. On the contrary, Gax stimulation inhibited AF proliferation and counteracted the effects of TGF-β1.
Effects of TGF-β1, Gax, and siRNA-Gax on AF Migration and Adhesion
By comparison, the values of migration length and adhesion ratio were significantly higher in the Ad-GFP+TGF-β1 group than those in the Ad-GFP group (supplemental Figure IVD and IVF), higher in the TGF-β1 and siRNA-Gax groups than those in the AF and siControl groups, higher in the siRNA-Gax+TGF-β1 group than those in the TGF-β1 and siRNA-Gax groups, and higher in the siRNA-Gax group than those in the TGF-β1 group (supplemental Figure IVE and IVG). These values were lower in the Ad-Gax+TGF-β1 and Ad-Gax groups than those in the Ad-GFP+TGF-β1 group. Thus, Gax knockdown more effectively stimulated AF migration and adhesion than TGF-β1 stimulation. On the contrary, Gax stimulation counteracted the effects of TGF-β1.
Effects of TGF-β1, Gax, and siRNA-Gax on AF Cell Cycle and Apoptosis
Compared with the Ad-GFP, AF, and siControl groups, the Ad-GFP+TGF-β1, TGF-β1, and siControl+TGF-β1 groups showed a insignificant trend of decreased AF number in G0-G1 phase and apoptosis, and a significant increase of AF number in S phase (Figure 3). Compared with the AF, TGF-β1, and siControl groups, the siRNA-Gax and siRNA-Gax+TGF-β1 groups displayed more AF cells in S and G2-M phases and less AF cells in G0-G1 phase and apoptosis (Figure 3B). In comparison with the Ad-GFP+TGF-β1, the Ad-Gax+TGF-β1 and Ad-Gax groups had significantly more AF cells in G0-G1 phase and apoptosis, and less AF cells in S phase (Figure 3A). Thus, TGF-β1 and siRNA-Gax increased the AF number in S phase, and Gax knockdown also increased G2-M proportion and decreased G0-G1 and apoptosis proportion in cell cycle. In contrast, Gax stimulation decreased the AF number in S phase and increased AF apoptosis.
Effects of TGF-β1, Gax, and siRNA-Gax on the Expression of Cell Cycle Regulators and Cytokines in Smad, Integrin, and MAPK Pathways
The mRNA expression of cell cycle regulator CDK4 and related cytokines in Smad, integrin, and MAPK pathways was increased and that of p15, p16, and p21 decreased in the Ad-GFP+TGF-β1, TGF-β1, and siRNA-Gax groups as compared with the Ad-GFP and AF groups (supplemental Figure VA). The siRNA-Gax+TGF-β1 group had higher mRNA expression of CDK4, Smads, integrins, and MAPKs, and lower mRNA expression of p15, p16, and p21 than the siRNA-Gax and TGF-β1 groups. The mRNA expression of CDK4, Smads, integrins, and MAPKs was reduced and that of p15, p16, and p21 was increased in the Ad-Gax+TGF-β1 and Ad-Gax groups compared with the Ad-GFP+TGF-β1 group (supplemental Figure VA and VB). Thus, TGF-β1 and siRNA-Gax upregulated the mRNA expression of CDK4, integrins, Smads, and MAPKs, and downregulated that of p15, p16, and p21. However, Gax stimulation counteracted the TGF-β1 effects.
The protein expression of CDK4, Smad, integrins, and MAPKs was upregulated but that of p16 or p21 was downregulated in the Ad-GFP+TGF-β1, TGF-β1, and siRNA-Gax groups as compared with the Ad-GFP, AF, and siControl groups (Figure 4, supplemental Figure VI). The protein expression of CDK4, Smads, integrins, and MAPKs was higher, while that of p16 and p21 was lower in the siRNA-Gax group was higher than that in the TGF-β1 group (Figure 4C, and 4D). The protein expression of CDK4, Smads, integrins, and MAPKs was decreased and that of p16 was increased in Ad-Gax+TGF-β1 and Ad-Gax groups as compared with the Ad-GFP+TGF-β1 group (Figure 4A and 4B; supplemental Figure VI). In particular, the protein expression of integrins showed a more marked change. These results suggest that TGF-β1 and siRNA-Gax upregulated the protein expression of CDK4, Smads, integrins, and MAPK pathways, and downregulated the expression of p16 and p21. However, Gax stimulation counteracted the TGF-β1 effects.
A body of evidence indicates that VECs, VSMCs, AF, and ECM are all involved in vascular proliferation. In particular, AF has been emphasized increasingly in vascular remodeling.1,2,20 At present, the mechanism of AF proliferation and migration is not yet clear. Moreover, few reports exist on gene interference in AF biofunction.
Among profibroblast growth factors, TGF-β1 is generally accepted as an important cytokine closely related to vascular remodeling.2–4 TGF-β inhibits the growth of most cells but stimulates growth of certain types of cells of mesenchymal-origin.21,22 The TGF-Smad pathway plays an important role in VECs and VSMCs proliferation.5,8,15 However, whether it has effect on AF remains unclear. Gene expression induced by TGF-β depends on Smad2, Smad3, or Smad4 in the Smad family.5,8,23 Our study showed that these factors all participate in AF proliferation process (supplemental Figure VA; Figure 4A and 4C; supplemental Figure VI). TGF-β regulates cell proliferation by affecting cell cycle dose- and time-dependently. Coexistence of other cytokines, cell density, and stages of cell growth all appear to modulate how TGF-β regulates cell cycle. Both positive regulation of the “engine device” and negative regulation of a “brake apparatus” are required for cycle regulation. Unlike specific CDK4-inhibiting factors p15 and p16 inactivating CDK4,24 nonspecific CDK inhibitor p21 inhibits most CDK activity.
Multifunctional cytokine TGF-β1 activates not only the Smad pathway but also non-Smad pathways,5,14,16,25,26 which cooperatively regulate the cell function.25–28 In fact, as transcription cofactors, Smads must interact with a specific DNA binding proteins to regulate gene transcription, so that other signaling coupling pathways may affect the TGF-Smad pathway at different levels.5,22,26 It is reported that the TGF-Smad pathway directly stimulates p38 and extracellular signal regulated kinase (ERK) pathways.6,8,14,16 The multifunction of TGF-β is attributable to the interactive effects of several signaling pathways.
As adhesion-independent pathways, Smad and MAPK pathways do not completely explain cell adhesion and migration phenomenon. The anchorage to ECM is essential for biofunction of many types of cells; reduction or removal of adhesion molecule matrix causes different degrees of apoptosis (anoikis).29,30 Integrin pathway is the main route influencing cell adhesion and migration. TGF-β1 regulates intercellular adhesion by remodeling ECM and activating FAK.30,31 Receptor-like integrin interacts extensively with other growth factors and their receptors.32 Moreover, Smads and integrins both activate MAPK pathways, so adhesion-independent Smad and adhesion-dependent integrin pathways may exhibit cross-talk.5 However, the specific mechanisms of integrin-mediated AF functional regulation remains unclear. Therefore, we presumed that the cross-talk between the Smad and integrin pathways may play a main role in mediating AF multifunction. TGF-β1 stimulation of AFs revealed that Smad expression increased and the expression of integrins and MAPKs simultaneously increased (Figure 4, supplemental Figure V and VI). After Gax intervention, Smads expression decreased and the expression of integrins and MAPKs also simultaneously decreased. In contrary, with siRNA-Gax treatment, the expressions of these signaling molecules were reversed, indicating that a cross-talk exists among Smad, MAPK, and integrin pathways in AF proliferation process. Therefore, these pathways interact and jointly constitute a molecular network in regulating AF cytobiological function.
As a “switch” for multiple intracellular signals, FAK activates many cytokines such as Ras and ERK to initiate a cascade of reactions and regulates cell function through intermolecular mechanisms.26–28,31,33 FAK-deficient mice showed a loss of mesenchymal cell motility and early embryonic lethality.7 PYK2, the important member of the FAK family, shares high homology with FAK and exerts an important effect on the regulation of cell functions.34 Whether PYK2 belongs to a subtype of FAK is still disputed. We showed PYK2 with the same change in mRNA and protein expression as FAK (supplemental Figure VB; Figure 4B and 4D), which suggests that both PYK2 and FAK act simultaneously on the same signaling pathway. Our findings support the hypothesis that PYK2 is not the subtype of FAK but the adhesion kinase of FAK downstream in the integrin pathway.35
Homeobox genes are critical in the processes of individual development, organogenesis, cell differentiation, and proliferation.36,37 Gax is the unique homeobox gene among repressing transcription factors. Unlike other cell cycle regulatory factors such as p21 and p53, the expression of Gax is largely confined to cardiovascular-specific and mesoderm-derived tissues. Gax can arrest the G0-to-G1 transition of cell cycle,9,38 and therefore Gax is at a switch position of regulating the cell cycle and may control more effectively cell proliferation.
Combining our results of AF bioactivity affected by Gax overexpression and knockdown with previous findings of Gax interference in VEC and VSMC function, we infer that the mechanism of Gax regulating cytobiological function is likely mediated by inhibiting angiocellular proliferation. In vascular proliferation, vascular cells express decreasing levels of Gax and vice versa. In vascular cells, the Gax overexpression upregulated p21 expression proportional to Gax dose.9,38 In our study, Gax upregulated not only p21, p15, and p16 expression but downregulated CDK4 expression. Therefore, Gax arrested the cell cycle before the G1 phase and effectively inhibited AF proliferation (Figure 3). Meanwhile, cell proliferation-related cytokines, such as FAK, activity can be regulated during the posttranslational period; however, the mechanism of transcriptional regulation is unclear. We hypothesize that the transcription regulator Gax may intervene in FAK transcription to regulate its activity. Unlike FAK-related nonkinase (FRNK),29 Gax may extensively regulate the transcription of many target genes rather than directionally intervene in a specific gene. Conversely, silencing endogenous Gax by siRNA upregulated expression of proliferative factors, downregulated expression of cell cycle inhibitors, and thus AFs proliferated. Therefore, Gax may attenuate the effects of TGF-β1 on cascade activation and signal amplification of Smad, MAPK, and integrin pathways, and at different degrees, inhibit the expression of related cytokines in these pathways, effectively coordinating cell signals.
Gax also inhibits angiocellular migration. It inhibits VSMC migration by downregulating integrinβ3 and β5 expression.11 In the present study, we found that Gax silencing and TGF-β1 upregulated integrin β1 expression, whereas Gax downregulated its expression in AFs. This finding suggests that integrin β1 may be the main subtype of integrin mediating AF adhesion and migration. Therefore, Gax may inhibit AF migration by blocking the integrin pathway. The third function of Gax is inducing angiocellular apoptosis. Previous studies demonstrated that Gax first induced cell cycle arrest and then induced VSMC apoptosis. We showed that Gax downregulated the expression of integrins, decreased AF proliferation and adhesion ability, and increased apoptosis. Thus, Gax may decrease the chance of AF survival and apoptosis escape by blocking integrin pathways and inhibiting AF adhesion. The inhibitory effects of Gax on AF proliferation were further confirmed by Gax silencing as demonstrated in this study.
In addition to aforementioned mechanisms, other potential mechanisms of Gax on cell bioactivity have been proposed. Gax may lead to upregulation of proapoptotic protein Bax and c-type natriuretic peptide and downregulation of antiapoptotic protein Bcl-2, NF-kB-dependent genes, c-myc, and angiotensin II, and thereby promote apoptosis, restrain cell growth, and inhibit angiogenic phenotype.10,13,39 Gax may suppress cell migration by inhibiting cell migratory response to platelet-derived growth factor-BB or hepatocyte growth factor.11
There were several limitations in this study. First, cultured rabbit AFs were treated with Gax plasmid derived from rat. The reason for not choosing rat AFs is because we planed to perform intravascular ultrasound (IVUS) studies to further validate our in vitro findings and rat is too small for imaging examinations. Second, anti-rabbit phosphorylation antibodies are not available and therefore the phosphorylation level of proteins in Smad, MAPK, and integrin signaling transduction pathways was not determined and further studies in this area are clearly warranted.
In summary, our data suggest that overlapping regulatory mechanisms of some signaling pathways could coordinate AF proliferation, migration, and adhesion. Conversely, at the transcription level, Gax can effectively inhibit the interaction of these pathways and interfere with AF biological functions. Although Gax can remarkably inhibit AF biofunction in vitro, whether Gax exerts favorable effects on vascular proliferation in vivo and the precise mechanisms of Gax interfering in AF bioactivity remain to be clarified.
We thank Prof Wei Cheng Hu and Zhe Yu Chen for their excellent advices.
Sources of Funding
This study was supported by the National 973 Basic Research Program of China (No. 2006CB503803), the National High-tech Research and Development Program of China (No. 2006AA02A406), and grants from the National Natural Science Foundation of China (No. 30470701, 30470702, 30570747, and 30670873).
P.L., C.Z., and J.B.F. contributed equally to this study.
Original received July 17, 2007; final version accepted December 31, 2007.
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