Focal Adhesion Kinase Degradation Research Articles

PDLIM7 Promotes Tumor Metastasis in Papillary Thyroid Carcinoma via Stabilizing Focal Adhesion Kinase Protein.

Background: As an actin cytoskeleton interactor, PDZ (postsynaptic density 65-discs large-zonula occludens 1) and LIM (abnormal cell lineage 11-isket 1-mechanosensory abnormal 3) domain protein 7 (PDLIM7) was supposed to play an essential role modulating cytoskeleton. Focal adhesions (FAs), which are located at the cytomembrane terminus of actin cytoskeleton, function as a force sensor and can transform the mechanical signal to a biochemical signal. Focal adhesion kinase (FAK) localizes to and regulates signal transduction in FAs, which play an essential role in cell polarity, migration, and invasion. However, whether PDLIM7 is involved in FAs-associated signal transduction and its role in tumor invasion and metastasis remains largely unknown. Methods: A cohort of 80 patients with papillary thyroid carcinoma (PTC) at The Second Affiliated Hospital of Guilin Medical University, as well as 512 PTC samples from The Cancer Genome Atlas thyroid cancer database was utilized to analyze the expression of PDLIM7 and its association with prognosis. Survival data were assessed using Kaplan-Meier curves, whereas clinicopathological characteristics such as age, sex, tumor size, multilocality, extrathyroidal extension, lymph metastases, Hashimoto's thyroiditis, distant metastasis, and TNM stage were considered. Functional assays were performed in vitro and in a xenograft mouse model to assess the role of PDLIM7 in PTC cell lines. The colocalization of PDLIM7 with FAK and integrin alpha V (ITGAV) was determined using immunofluorescence assay and immunoprecipitation assay. Protein expression levels in cell and tissue biopsies were measured through Western blotting and immunohistochemistry. Results: (1) The PDLIM7 protein frequently upregulated in both PTC tissues and cells, and overexpression of PDLIM7 is associated with advanced clinicopathological characteristics. (2) Knockdown of PDLIM7 effectively inhibits cell proliferation, migration, and invasion in PTC cell lines in vitro. (3) Knockdown of PDLIM7 hinders the growth and metastasis of TPC-1 xenografts in vivo. (4) PDLIM7 demonstrates colocalization with both FAK and the FA molecule ITGAV and the knockdown of PDLIM7 resulted in disassembly of FAs and proteosome-dependent degradation of FAK in PTC cell lines. Conclusions: PDLIM7 function as an oncoprotein in PTC to promote metastasis, and a novel underlying mechanism is proposed that PDLIM7 keeps FAK protein from proteosome-dependent degradation.

Degradation of FAK-targeting by proteolytic targeting chimera technology to inhibit the metastasis of hepatocellular carcinoma.

Liver cancer is a prevalent malignant cancer, ranking third in terms of mortality rate. Metastasis and recurrence primarily contribute to the high mortality rate of liver cancer. Hepatocellular carcinoma (HCC) has low expression of focal adhesion kinase (FAK), which increases the risk of metastasis and recurrence. Nevertheless, the efficacy of FAK phosphorylation inhibitors is currently limited. Thus, investigating the mechanisms by which FAK affects HCC metastasis to develop targeted therapies for FAK may present a novel strategy to inhibit HCC metastasis. This study examined the correlation between FAK expression and the prognosis of HCC. Additionally, we explored the impact of FAK degradation on HCC metastasis through wound healing experiments, transwell invasion experiments, and a xenograft tumor model. The expression of proteins related to epithelial-mesenchymal transition (EMT) was measured to elucidate the underlying mechanisms. The results showed that FAK PROTAC can degrade FAK, inhibit the migration and invasion of HCC cells in vitro, and notably decrease the lung metastasis of HCC in vivo. Increased expression of E-cadherin and decreased expression of vimentin indicated that EMT was inhibited. Consequently, degradation of FAK through FAK PROTAC effectively suppressed liver cancer metastasis, holding significant clinical implications for treating liver cancer and developing innovative anti-neoplastic drugs.

Development and Characterization of Selective FAK Inhibitors and PROTACs with In Vivo Activity.

Focal adhesion kinase (FAK) is an attractive drug target due to its overexpression in cancer. FAK functions as a non-receptor tyrosine kinase and scaffolding protein, coordinating several downstream signaling effectors and cellular processes. While drug discovery efforts have largely focused on targeting FAK kinase activity, FAK inhibitors have failed to show efficacy as single agents in clinical trials. Here, using structure-guided design, we report the development of a selective FAK inhibitor (BSJ-04-175) and degrader (BSJ-04-146) to evaluate the consequences and advantages of abolishing all FAK activity in cancer models. BSJ-04-146 achieves rapid and potent FAK degradation with high proteome-wide specificity in cancer cells and induces durable degradation in mice. Compared to kinase inhibition, targeted degradation of FAK exhibits pronounced improved activity on downstream signaling and cancer cell viability and migration. Together, BSJ-04-175 and BSJ-04-146 are valuable chemical tools to dissect the specific consequences of targeting FAK through small-molecule inhibition or degradation.

FAK suppresses antigen processing and presentation to promote immune evasion in pancreatic cancer

ObjectiveImmunotherapy for the treatment of pancreatic ductal adenocarcinoma (PDAC) has shown limited efficacy. Poor CD8 T-cell infiltration, low neoantigen load and a highly immunosuppressive tumour microenvironment contribute to this lack...

Identification of novel and potent PROTACs targeting FAK for non-small cell lung cancer: Design, synthesis, and biological study

The intracellular non-receptor tyrosine protein kinase Focal adhesion kinase (FAK) is a key signalling regulator, which mediates tumor survival, invasion, metastasis, and angiogenesis through its kinase catalytic functions and non-kinase scaffolding functions. Previous efforts have clarified that it is crucial to address both FAK kinase and scaffolding functions instead of just inhibiting FAK kinase activity because it may be insufficient to completely block FAK signaling. Proteolysis targeting chimera (PROTAC) technology is a method of targeting a specific protein and inducing its degradation in the cell, which can simultaneously eliminate both kinase-dependent enzymatic functions and scaffolding functions. In current study, we designed and synthesized a series of novel FAK PROTACs and the optimal PROTAC B5 exhibited potent FAK affinity with an IC50 value of 14.9 nM. Furthermore, in A549 cells, PROTAC B5 presented strong FAK degradation activity (86.4% degradation @ 10 nM), powerful antiproliferative activity (IC50 = 0.14 ± 0.01 μM) and inhibited cell migration and invasion in a concentration-dependent manner. Additionally, the in vitro preliminary drug-like properties evaluation of PROTAC B5 showed outstanding plasma stability and moderate membrane permeability. Together, current results provided a promising FAK PROTAC B5 as lead compound for cancer-related drug discovery and FAK-degradation functions exploration in biological systems.

Discovery and Characterisation of Highly Cooperative FAK-Degrading PROTACs.

Focal adhesion kinase (FAK) is a key mediator of tumour progression and metastasis. To date, clinical trials of FAK inhibitors have reported disappointing efficacy for oncology indications. We report the design and characterisation of GSK215, a potent, selective, FAK-degrading Proteolysis Targeting Chimera (PROTAC) based on a binder for the VHL E3 ligase and the known FAK inhibitor VS-4718. X-ray crystallography revealed the molecular basis of the highly cooperative FAK-GSK215-VHL ternary complex, and GSK215 showed differentiated in-vitro pharmacology compared to VS-4718. In mice, a single dose of GSK215 induced rapid and prolonged FAK degradation, giving a long-lasting effect on FAK levels (≈96 h) and a marked PK/PD disconnect. This tool PROTAC molecule is expected to be useful for the study of FAK-degradation biology in vivo, and our results indicate that FAK degradation may be a differentiated clinical strategy versus FAK inhibition for the treatment of cancer.

Induction of cyclophilin A by influenza A virus infection facilitates group A Streptococcus coinfection.

During influenza A epidemics, bacterial coinfection is a major cause of increased morbidity and mortality. However, the roles of host factors in regulating influenza A virus (IAV)-triggered bacterial coinfection remain elusive. Cyclophilin A (CypA) is an important regulator of infection and immunity. Here, we show that IAV-induced CypA expression facilitates group A Streptococcus (GAS) coinfection both invitro and invivo. Upon IAV infection, CypA interacts with focal adhesion kinase (FAK) and inhibited E3 ligase cCbl-mediated, K48-linked ubiquitination of FAK, which positively regulates integrin α5 expression and actin rearrangement via the FAK/Akt signaling pathway to facilitate GAS colonization and invasion. Notably, CypA deficiency or inhibition by cyclosporine A significantly inhibits IAV-triggered GAS coinfection in mice. Collectively, thesefindings reveal that CypA is critical for GAS infection, and induction of CypA expression is another way for IAV to promote bacterial coinfection, suggesting that CypA is a promising therapeutic target for the secondary bacterial infection.

Progress in the Development of Small Molecular Inhibitors of Focal Adhesion Kinase (FAK)

Focal adhesion kinase (FAK) is a nonreceptor intracellular tyrosine kinase that plays an essential role in cancer cell adhesion, survival, proliferation, and migration through both its enzymatic activities and scaffolding functions. Overexpression of FAK has been found in many human cancer cells from different origins, which promotes tumor progression and influences clinical outcomes in different classes of human tumors. Therefore, FAK has been considered as a promising target for small molecule anticancer drug development. Many FAK inhibitors targeting different domains of FAK with various mechanisms of functions have been reported, including kinase domain inhibitors, FERM domain inhibitors, and FAT domain inhibitors. In addition, FAK-targeting PROTACs, which can induce the degradation of FAK, have also been developed. In this Perspective, we summarized the progress in the development of small molecular FAK inhibitors and proposed the perspectives for the future development of agents targeting FAK.

Design, Synthesis, and Evaluation of Highly Potent FAK-Targeting PROTACs.

Focal adhesion kinase (FAK), a cytoplasmic protein tyrosine kinase, exerts kinase-dependent enzymatic functions and kinase-independent scaffolding functions, both of which are crucial in cancer development, early embryonic development, and reproduction. However, previous efforts for FAK blocking mainly focus on kinase inhibitors. Proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules that allow direct post-translational knockdown of proteins via ubiquitination of a target protein by E3 ubiquitin ligase and subsequent proteasomal degradation. Here, we designed and synthesized a FAK PROTAC library with FAK inhibitor (PF562271 or VS6063) and CRBN E3 ligand. A novel FAK-targeting PROTAC, FC-11, showed a rapid and reversible FAK degradation with a picomolar of DC50 in various cell lines in vitro, which imply that FAK-PROTACs could be useful as expand tools for studying functions of FAK in biological system and as potential therapeutic agents.

Histidine-rich glycoprotein ameliorates endothelial barrier dysfunction through regulation of NF-κB and MAPK signal pathway.

Background and PurposeMicrovascular barrier breakdown is a hallmark of sepsis that is associated with sepsis‐induced multiorgan failure. Histidine‐rich glycoprotein (HRG) is a 75‐kDa plasma protein that was demonstrated to improve the survival of septic mice through regulation of cell shape, spontaneous ROS production in neutrophils, and adhesion of neutrophils to vascular endothelial cells. We investigated HRG's role in the LPS/TNF‐α‐induced barrier dysfunction of endothelial cells in vitro and in vivo and the possible mechanism, to clarify the definitive roles of HRG in sepsis.Experimental ApproachEA.hy 926 endothelial cells were pretreated with HRG or human serum albumin before stimulation with LPS/TNF‐α. A variety of biochemical assays were applied to explore the underlying molecular mechanisms on how HRG protected the barrier function of vascular endothelium.Key ResultsImmunostaining results showed that HRG maintains the endothelial monolayer integrity by inhibiting cytoskeleton reorganization, losses of VE‐cadherin and β‐catenin, focal adhesion kinase degradation, and cell detachment induced by LPS/TNF‐α. HRG also inhibited the cytokine secretion from endothelial cells induced by LPS/TNF‐α, which was associated with reduced NF‐κB activation. Moreover, HRG effectively prevented the LPS/TNF‐α‐induced increase in capillary permeability in vitro and in vivo. Finally, Western blot results demonstrated that HRG prevented the phosphorylation of MAPK family and RhoA activation, which are involved mainly in the regulation of cytoskeleton reorganization and barrier permeability.Conclusions and ImplicationsTaken together, our results demonstrate that HRG has protective effects on vascular barrier function in vitro and in vivo, which may be due to the inhibition of MAPK family and Rho activation.

Cbl-b promotes cell detachment via ubiquitination of focal adhesion kinase.

Cancer cell detachment from the primary tumor site represents the first stage of metastasis. Previous studies have identified that cell detachment is triggered by cytoskeletal disruption, which may induce a wide variety of cellular changes. Focal adhesion kinase (FAK) exhibits crucial cellular functions, including regulation of the cytoskeleton. These observations have provided exciting insights into the effect of FAK in cell detachment; however, the involvement of FAK in cell detachment remains controversial. The aim of the present study was to evaluate the effect of FAK and its function in the process of cell detachment. The results revealed that FAK expression was downregulated following trypsin treatment in human gastric, lung, colon and breast cancer cell lines, as well as a human gastric epithelial cell line. Knockdown of FAK enhanced cell detachment in gastric cancer MGC803 cells, indicating that FAK inhibits cell detachment. Further investigation revealed that trypsin induced monoubiquitination of FAK. In addition, the lysosome inhibitor, NH4Cl, decreased trypsin-induced degradation of FAK. Casitas B-lineage lymphoma-b (Cbl-b), an E3 ubiquitin ligase, was involved in this process, which interacted with FAK, as demonstrated by co-precipitation experiments, and promoted trypsin-induced ubiquitin-lysosome degradation of FAK. These results indicate that Cbl-b promotes cell detachment via ubiquitination of FAK. These findings provide novel insights regarding the effect of FAK and Cbl-b in the process of cancer cell detachment.

Focal adhesion kinase signaling is decreased 56 days following spinal cord injury in rat gastrocnemius

Descriptive study. The goal of this study was to determine the effects of spinal cord injury (SCI) on aspects of the focal adhesion kinase (FAK) signaling pathway 56 days post injury in rat gastrocnemius. This study was conducted in Bronx, NY, USA. Three-month-old male Wistar rats were exposed to either a sham surgery (n=10) or complete T4 spinal cord transection (n=10). Rats were killed 56 days following surgery and the muscle was collected. Following homogenization, proteins of the FAK pathway were analyzed by western immunoblotting or reverse transcription-qPCR. In addition, cellular markers for proteins that target the degradation of FAK were investigated. SCI resulted in significantly lower levels of total and phosphorylated FAK, cSrc and p70S6k, and a trend for increased FRNK protein expression. SCI did not change levels of the α7 or β1 integrin subunits, total or phosphorylated ERK1/2, phosphorylated Akt and TSC2 or total p70S6k. SCI resulted in a greater expression of total Akt. mRNA expression of FAK and the α7 or β1 integrins remained unchanged between sham and SCI groups. Caspase-3/7 activity and Trim72 mRNA and protein expression remained unchanged following SCI. SCI results in diminished FAK signaling and is independent of ERK1/2 and Akt. SCI has no effect on mRNA levels for genes encoding components of the focal adhesion 56 days after injury.

Impaired Focal Adhesion Kinase-Grb2 Interaction during Elevated Activity in Hippocampal Neurons.

Excitatory/inhibitory imbalances are implicated in many neurological disorders. Previously, we showed that chronically elevated network activity induces vulnerability in neurons due to loss of signal transducer and activator of transcription 3 (STAT3) signaling in response to the impairment of the serine/threonine kinase, extracellular-signal-regulated kinases 1/2 (Erk1/2) activation. However, how phosphorylation of Erk1/2 decreases during elevated neuronal activity was unknown. Here I show the pErk1/2 decrease induced by 4-aminopyridine (4-AP), an A-type potassium channel inhibitor can be blocked by a broad-spectrum matrix-metalloproteinase (MMP) inhibitor, FN-439. Surface expression levels of integrin β1 dramatically decrease when neurons are challenged by chronically elevated activity, which is reversed by FN-439. Treatment with 4-AP induces degradation of focal adhesion kinase (FAK), the mediator of integrin signaling. As a result, interactions between FAK and growth factor receptor-bound protein 2 (Grb2), the adaptor protein that mediates Erk1/2 activation by integrin, are severely impaired. Together, these data suggest the loss of integrin signaling during elevated activity causes vulnerability in neurons.

αB-crystallin interacts with and prevents stress-activated proteolysis of focal adhesion kinase by calpain in cardiomyocytes.

Focal adhesion kinase (FAK) contributes to cellular homeostasis under stress conditions. Here we show that αB-crystallin interacts with and confers protection to FAK against calpain-mediated proteolysis in cardiomyocytes. A hydrophobic patch mapped between helices 1 and 4 of the FAK FAT domain was found to bind to the β4-β8 groove of αB-crystallin. Such an interaction requires FAK tyrosine 925 and is enhanced following its phosphorylation by Src, which occurs upon FAK stimulation. αB-crystallin silencing results in calpain-dependent FAK depletion and in the increased apoptosis of cardiomyocytes in response to mechanical stress. FAK overexpression protects cardiomyocytes depleted of αB-crystallin against the stretch-induced apoptosis. Consistently, load-induced apoptosis is blunted in the hearts from cardiac-specific FAK transgenic mice transiently depleted of αB-crystallin by RNA interference. These studies define a role for αB-crystallin in controlling FAK function and cardiomyocyte survival through the prevention of calpain-mediated degradation of FAK.

EspC promotes epithelial cell detachment by enteropathogenic Escherichia coli via sequential cleavages of a cytoskeletal protein and then focal adhesion proteins.

EspC is a non-locus of enterocyte effacement (LEE)-encoded autotransporter produced by enteropathogenic Escherichia coli (EPEC) that is secreted to the extracellular milieu by a type V secretion system and then translocated into epithelial cells by the type III secretion system. Here, we show that this efficient EspC delivery into the cell leads to a cytopathic effect characterized by cell rounding and cell detachment. Thus, EspC is the main protein involved in epithelial cell cytotoxicity detected during EPEC adhesion and pedestal formation assays. The cell detachment phenotype is triggered by cytoskeletal and focal adhesion disruption. EspC-producing EPEC is able to cleave fodrin, paxillin, and focal adhesion kinase (FAK), but these effects are not observed when cells are infected with an espC isogenic mutant. Recovery of these phenotypes by complementing the mutant with the espC gene but not with the espC gene mutated in the serine protease motif highlights the role of the protease activity of EspC in the cell detachment phenotype. In vitro assays using purified proteins showed that EspC, but not EspC with an S256I substitution [EspCS256I], directly cleaves these cytoskeletal and focal adhesion proteins. Kinetics of protein degradation indicated that EspC-producing EPEC first cleaves fodrin (within the 11th and 9th repetitive units at the Q1219 and D938 residues, respectively), and this event sequentially triggers paxillin degradation, FAK dephosphorylation, and FAK degradation. Thus, cytoskeletal and focal adhesion protein cleavage leads to the cell rounding and cell detachment promoted by EspC-producing EPEC.

Saikosaponin C inhibits lipopolysaccharide-induced apoptosis by suppressing caspase-3 activation and subsequent degradation of focal adhesion kinase in human umbilical vein endothelial cells

Bacterial lipopolysaccharide (LPS) is an important mediator of inflammation and a potent inducer of endothelial cell damage and apoptosis. In this study, we investigated the protective effects of saikosaponin C (SSc), one of the active ingredients produced by the traditional Chinese herb, Radix Bupleuri, against LPS-induced apoptosis in human umbilical endothelial cells (HUVECs). LPS triggered caspase-3 activation, which was found to be important in LPS-induced HUVEC apoptosis. Inhibition of caspase-3 also inhibited LPS-induced degradation of focal adhesion kinase (FAK), indicating that caspase-3 is important in LPS-mediated FAK degradation as well as in apoptosis in HUVECs. SSc significantly inhibited LPS-induced apoptotic cell death in HUVECs through the selective suppression of caspase-3. SSc was also shown to rescue LPS-induced FAK degradation and other cell adhesion signals. Furthermore, the protective effects of SSc against LPS-induced apoptosis were abolished upon pretreatment with a FAK inhibitor, highlighting the importance of FAK in SSc activity. Taken together, these results show that SSc efficiently inhibited LPS-induced apoptotic cell death via inhibition of caspase-3 activation and caspase-3-mediated-FAK degradation. Therefore, SSc represents a promising therapeutic candidate for the treatment of vascular endothelial cell injury and cellular dysfunction.

Mitsugumin 53 (MG53) Ligase Ubiquitinates Focal Adhesion Kinase during Skeletal Myogenesis

The striated muscle-specific mitsugumin 53 (MG53) is a novel E3 ligase that induces the ubiquitination of insulin receptor substrate 1 (IRS-1) during skeletal myogenesis, negatively regulating insulin-like growth factor and insulin signaling. Here we show that focal adhesion kinase (FAK) is the second target of MG53 during skeletal myogenesis. The FAK protein level gradually decreased, whereas its mRNA level was constant during myogenesis in C2C12 cells and MyoD-overexpressing mouse embryonic fibroblasts. The FAK protein was associated with the E2 enzyme UBE2H and the E3 enzyme MG53 in endogenous and exogenous immunoprecipitation experiments. FAK ubiquitination and degradation was induced by MG53 overexpression in myoblasts but abolished by MG53 or UBE2H knockdown in myotubes. Because RING-disrupted MG53 mutants (C14A and ΔR) did not induce FAK ubiquitination and degradation, the RING domain was determined to be required for MG53-induced FAK ubiquitination. Taken together, these data indicate that MG53 induces FAK ubiquitination with the aid of UBE2H during skeletal myogenesis.

Binding of a Sialic Acid-recognizing Lectin Siglec-9 Modulates Adhesion Dynamics of Cancer Cells via Calpain-mediated Protein Degradation

Although regulatory mechanisms for immune cells with inhibitory signals via immunoreceptor tyrosine-based inhibitory motifs are well known, signals transduced via interaction between Siglecs and sialyl compounds on their counterreceptors into target cells have not been reported to date. In this study, we found that an astrocytoma cell line, AS, showed detachment from culture plates when co-cultured with Siglec-9-expressing cells and/or soluble Siglec-9. Moreover, detached AS cells regrew as co-cultured cells with Siglec-9-deficient cells. They also showed increased motility and invasiveness upon Siglec-9 binding. In immunoblotting, rapid degradation of focal adhesion kinase (FAK) and related signaling molecules such as Akt, paxillin, and p130Cas was observed immediately after the co-culture. Despite degradation of these molecules, increased p-Akt was found at the front region of the cytoplasm, probably reflecting increased cell motility. Calpain was considered to be a responsible protease for the protein degradation by the inhibition experiments. These results suggest that protein degradation of FAK and related molecules was induced by Siglec-9 binding to its counterreceptors via sialylglycoconjugates, leading to the modulation of adhesion kinetics of cancer cells. Thus, this might be a mechanism by which cancer cells utilize Siglec-9-derived signals to escape from immunosurveillance.

Progesterone receptor isoforms PRA and PRB differentially contribute to breast cancer cell migration through interaction with focal adhesion kinase complexes

Progesterone receptor (PR) and progestins affect mammary tumorigenesis; however, the relative contributions of PR isoforms A and B (PRA and PRB, respectively) in cancer cell migration remains elusive. By using a bi-inducible MDA-MB-231 breast cancer cell line expressing PRA and/or PRB, we analyzed the effect of conditional PR isoform expression. Surprisingly, unliganded PRB but not PRA strongly enhanced cell migration as compared with PR(-) cells. 17,21-Dimethyl-19-norpregna-4,9-dien-3,20-dione (R5020) progestin limited this effect and was counteracted by the antagonist 11β-(4-dimethyl-amino)-phenyl-17β-hydroxy-17-(1-propynyl)-estra-4,9-dien-3-one (RU486). Of importance, PRA coexpression potentiated PRB-mediated migration, whereas PRA alone was ineffective. PR isoforms differentially regulated expressions of major players of cell migration, such as urokinase plasminogen activator (uPA), its inhibitor plasminogen activator inhibitor type 1, uPA receptor (uPAR), and β1-integrin, which affect focal adhesion kinase (FAK) signaling. Moreover, unliganded PRB but not PRA enhanced FAK Tyr397 phosphorylation and colocalized with activated FAK in cell protrusions. Because PRB, as well as PRA, coimmunoprecipitated with FAK, both isoforms can interact with FAK complexes, depending on their respective nucleocytoplasmic trafficking. In addition, FAK degradation was coupled to R5020-dependent turnovers of PRA and PRB. Such an effect of PRB/PRA expression on FAK signaling might thus affect adhesion/motility, underscoring the implication of PR isoforms in breast cancer invasiveness and metastatic evolution with underlying therapeutic outcomes.

Mechanical Stress Modulates SOCS-1 Expression in Human Vascular Smooth Muscle Cells

Background: The growth-promoting effect of mechanical stress on vascular smooth muscle cells (VSMC) has been implicated in the progress of cardiovascular diseases related to elevated blood pressure. The underlying molecular mechanisms are, however, not completely defined. Methods: We have studied primary human aortic VSMC using a model for multilateral stretch. Expression of the suppressor of cytokine signaling (SOCS) family member SOCS-1 and related molecular mechanisms were studied using TaqMan analysis, immunoblotting, protein silencing, specific cell treatment, immunoprecipitation and immunocytochemistry. Results: Mechanical stretch inhibits SOCS-1 mRNA and protein expression. This effect was abolished by cell treatment with methyl-β-cyclodextrin disrupting lipid rafts and with RGD peptide affecting integrins. Inhibition of integrin interaction with another cellular receptor, urokinase receptor (uPAR), as well as uPAR silencing also abolished stretch-induced SOCS-1 downregulation. Mechanical stretch resulted in uPAR redistribution to lipid rafts and in its colocalization with focal adhesion kinase (FAK). Stretch impairs polyubiquitination and proteosomal degradation of FAK leading to FAK upregulation in stretched VSMC. SOCS-1 silencing and inhibition of proteosomal degradation simulate this effect. Conclusion: Our study identifies SOCS-1 as a novel participant involved in the propagation of mechanical stimuli in human VSMC, which might be relevant for the development of cardiovascular diseases.