Caveolin-1 Scaffolding Domain Research Articles

Synthetic cell-permeable caveolin-1 scaffolding domain peptide activates phagocytosis of Escherichia coli by regulating Rab5 activity.

Caveolae are defined as 50-100nm wide pits in the plasma membrane containing oligomeric caveolin proteins. They have been implicated in endocytosis (including phagocytosis), transcytosis, calcium signalling, and numerous other signal transduction events. Caveolin-1, a major structural component of caveolae, enhances Rab5 activity. In this study, we examined the effect of a synthetic cell-permeable peptide of the caveolin-1 scaffolding domain (CSD) on phagocytosis. Treatment with the CSD peptide increased Rab5 activity, Rab5-early endosome antigen 1 (EEA1) interaction, and phagocytosis of Escherichia coli. The results suggest that the synthetic cell-permeable CSD peptide is an activator of phagocytosis.

Tyrosine phosphorylation of tumor cell caveolin-1: impact on cancer progression.

Caveolin-1 (CAV1) has long been implicated in cancer progression, and while widely accepted as an oncogenic protein, CAV1 also has tumor suppressor activity. CAV1 was first identified in an early study as the primary substrate of Src kinase, a potent oncoprotein, where its phosphorylation correlated with cellular transformation. Indeed, CAV1 phosphorylation on tyrosine-14 (Y14; pCAV1) has been associated with several cancer-associated processes such as focal adhesion dynamics, tumor cell migration and invasion, growth suppression, cancer cell metabolism, and mechanical and oxidative stress. Despite this, a clear understanding of the role of Y14-phosphorylated pCAV1 in cancer progression has not been thoroughly established. Here, we provide an overview of the role of Src-dependent phosphorylation of tumor cell CAV1 in cancer progression, focusing on pCAV1 in tumor cell migration, focal adhesion signaling and metabolism, and in the cancer cell response to stress pathways characteristic of the tumor microenvironment. We also discuss a model for Y14 phosphorylation regulation of CAV1 effector protein interactions via the caveolin scaffolding domain.

Caveolin-1 Scaffolding Domain Peptide Regulates Colon Endothelial Cell Survival through JNK Pathway.

It has been reported that pathological angiogenesis contributes to both experimental colitis and inflammatory bowel disease. Recently, we demonstrated that endothelial caveolin-1 plays a key role in the pathological angiogenesis of dextran sodium sulfate (DSS) colitis. However, the molecular mechanism of caveolin-1 regulation of endothelial function is unknown. In this study, we examined how the antennapedia- (AP-) conjugated caveolin-1 scaffolding domain (AP-Cav) modulates vascular endothelial growth factor- (VEGF-) dependent colon endothelial cell angiogenic responses, as seen during colitis. We used mouse colon endothelial cells and found that AP-Cav significantly inhibited VEGF-mediated bromodeoxyuridine (BrdU) incorporation into colon microvascular endothelial cells. AP-Cav significantly blunted VEGF-dependent extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation at 10 minutes and 2 hours after stimulation, compared with the AP control peptide. AP-Cav + VEGF-A treatment also significantly increased c-Jun N-terminal kinase (JNK) phosphorylation at 2 hours. AP-Cav + VEGF-A treatment significantly downregulated retinoblastoma (Rb) protein levels, upregulated cleaved caspase-3 protein levels at 4 hours, and induced apoptosis. Thus, our study suggests that disruption of endothelial caveolin-1 function via the AP-Cav diverts VEGF signaling responses away from endothelial cell proliferation and toward apoptosis through the inhibition of mitogen-activated protein (MAP) kinase signaling and the induction of JNK-associated apoptosis.

Caveolin-1 alleviates lipid accumulation in NAFLD associated with promoting autophagy by inhibiting the Akt/mTOR pathway

Non-alcoholic fatty liver disease (NAFLD) is the most burgeoning chronic liver disease worldwide whose pathogenesis is complex and controversial. Here, we investigated the impact of caveolin-1 (CAV1), a scaffolding protein of caveolae for lipid homeostasis and endocytosis, on the pathogenesis of NAFLD. CAV1 and caveolae play crucial roles in the regulation of autophagy and hepatic energy metabolism. However, it remains unclear whether CAV1 could affect hepatic lipid metabolism by regulating autophagy. In this study, results showed that the expressions of CAV1 and autophagy-related proteins (Beclin1 and LC3-II/Ⅰ) were decreased, while the level of p62 was increased in HFD (high-fat diet) fed mice liver and in A/O (alcohol and oleic acid mixture) treated L02 cells, compared to the corresponding controls. In vivo study, upregulation of CAV1 with CAV1 scaffolding domain peptides (CSD, amino acids 82–101 of caveolin-1) could alleviate lipid accumulation and promote autophagy in NAFLD mice. In vitro study, CAV1 overexpression plasmid and its small interfering RNA were cultured with A/O treated L02 cells respectively. The results also demonstrated that CAV1 reduced lipid accumulation and promoted autophagy in L02 cells. Treatment with chloroquine, an inhibitor of autophagic degradation, abrogated CAV1 plasmid-mediated alleviation of lipid accumulation. Mechanistically, the inhibition of Akt/mTOR pathway was involved in the protective role of CAV1 in autophagy induction and lipid metabolism in NAFLD. Together, these results provided novel perception into the function of CAV1 in liver through autophagy and emphasized its positive role in NAFLD.

Development of an Excipient-Free Peptide Dry Powder Inhalation for the Treatment of Pulmonary Fibrosis.

The caveolin scaffolding domain peptide (CSP) is being developed for the therapeutic intervention of a lethal lung disease, idiopathic pulmonary fibrosis. While direct respiratory delivery of CSP7 (a 7-mer fragment of CSP) is considered an effective route, proper formulation and processing of the peptide are required. First, air-jet milling technology was performed in order to micronize the neat peptide powder. Next, the fine particles were subjected to a stability study with physical and chemical characterizations. In addition, the in vivo efficacy of processed CSP7 powder was evaluated in an animal model of lung fibrosis. The results revealed that, with jet milling, the particle size of CSP7 was reduced to a mass median aerodynamic diameter of 1.58 ± 0.1 μm and 93.3 ± 3.3% fine particle fraction, optimal for deep lung delivery. A statistically significant reduction of collagen was observed in diseased lung tissues of mice that received CSP7 powder for inhalation. The particles remained chemically and physically stable after micronization and during storage. This work demonstrated that jet milling is effective in the manufacturing of a stable, excipient-free CSP7 inhalation powder for the treatment of pulmonary fibrosis.

Formulation Composition and Process Affect Counterion for CSP7 Peptide.

Counterions commonly remain with peptides in salt form after peptide purification. In animal and human studies, acetate counterions are a safer and more acceptable choice for peptides than others (e.g., trifluoroacetate counterions). Various salt forms of caveolin-1 scaffolding domain (CSP7) affect counterion volatilization. The development of lyophilized formulations containing volatile compounds is a challenge because these compounds sublimate away during the process. This work aims to investigate the effect of excipients and lyophilization parameters on the preservation of volatile compounds after lyophilization. The peak areas obtained from 1H and 19F NMR spectra were used to calculate the molar ratio of counterions to CSP7. We found that the pH modifier excipient had the greatest impact on the loss of counterions. By optimizing the molar ratio of bulking agent to CSP7, volatile compounds can be preserved after lyophilization. Higher chamber pressure during lyophilization can lower the sublimation rate of volatile compounds. Moreover, the loss of volatile compounds affects the stability of CSP7 due to the pH shift of reconstituted solutions, thereby causing peptide aggregation. The optimization of the formulation and processing helps preserve volatile compounds, thus minimizing the pH change of reconstituted solutions and maintaining the stability of peptide.

Deletion of caveolin scaffolding domain alters cancer cell migration

ABSTRACTCaveolin-1 (Cav-1) is an integral membrane protein that plays an important role in proliferative and terminally differentiated cells. As a structural component of Caveolae, Cav-1 interacts with signaling molecules via a caveolin scaffolding domain (CSD) regulating cell signaling. Recent reports have shown that Cav-1 is a negative regulator in tumor metastasis. Therefore, we hypothesize that Cav-1 inhibits cell migration through its CSD. HeLa cells were engineered to overexpress Cav-1 (Cav-1 OE), Cav-1 without a functional CSD (∆CSD), or enhanced green fluorescent protein (EGFP) as a control. HeLa cell migration was suppressed in Cav-1 OE cells while ∆CSD showed increased migration, which corresponded to a decrease in the tight junction protein, zonula occludens (ZO-1). The migration phenotype was confirmed in multiple cancer cell lines. Phosphorylated STAT-3 was decreased in Cav-1 OE cells compared to control and ∆CSD cells; reducing STAT-3 expression alone decreased cell migration. ∆CSD blunted HeLa proliferation by increasing the number of cells in the G2/M phase of the cell cycle. Overexpressing the CSD peptide alone suppressed HeLa cell migration and inhibited pSTAT3. These findings suggest that Cav-1 CSD may be critical in controlling the dynamic phenotype of cancer cells by facilitating the interaction of specific signal transduction pathways, regulating STAT3 and participating in a G2/M checkpoint. Modulating the CSD and targeting specific proteins may offer potential new therapies in the treatment of cancer metastasis.

Caveolin-1 scaffolding domain peptide prevents hyperoxia-induced airway remodeling in a neonatal mouse model.

Reactive airway diseases are significant sources of pulmonary morbidity in neonatal and pediatric patients. Supplemental oxygen exposure in premature infants contributes to airway diseases such as asthma and promotes development of airway remodeling, characterized by increased airway smooth muscle (ASM) mass and extracellular matrix (ECM) deposition. Decreased plasma membrane caveolin-1 (CAV1) expression has been implicated in airway disease and may contribute to airway remodeling and hyperreactivity. Here, we investigated the impact of clinically relevant moderate hyperoxia (50% O2) on airway remodeling and caveolar protein expression in a neonatal mouse model. Within 12 h of birth, litters of B6129SF2J mice were randomized to room air (RA) or 50% hyperoxia exposure for 7 days with or without caveolin-1 scaffolding domain peptide (CSD; caveolin-1 mimic; 10 µl, 0.25 mM daily via intraperitoneal injection) followed by 14 days of recovery in normoxia. Moderate hyperoxia significantly increased airway reactivity and decreased pulmonary compliance at 3 wk. Histologic assessment demonstrated airway wall thickening and increased ASM mass following hyperoxia. RNA from isolated ASM demonstrated significant decreases in CAV1 and cavin-1 in hyperoxia-exposed animals while cavin-3 was increased. Supplementation with intraperitoneal CSD mitigated both the physiologic and histologic changes observed with hyperoxia. Overall, these data show that moderate hyperoxia is detrimental to developing airway and may predispose to airway reactivity and remodeling. Loss of CAV1 is one mechanism through which hyperoxia produces these deleterious effects. Supplementation of CAV1 using CSD or similar analogs may represent a new therapeutic avenue for blunting hyperoxia-induced pulmonary damage in neonates.

Correction: Lu, J. et al. Caveolin-1 Scaffolding Domain Peptides Alleviate Liver Fibrosis by Inhibiting TGF-β1/Smad Signaling in Mice. Int. J. Mol. Sci., 2018, 19, 1729.

The author wishes to make the following correction to this paper [...].

Caveolin scaffolding domain plays an important role in cancer cell migration

Caveolin‐1 is the integral membrane protein that plays an important role in the structure and function of terminally differentiated cells. As a structural component of caveolae, the caveolin scaffolding domain (CSD) has been associated with many cellular signaling processes and recent reports have implicated Cav‐1 as a negative regulator in tumor metastasis. We hypothesized that Cav‐1 may be inhibiting cell migration through its CSD. To assess this, HCT‐116 cells (which contain high endogenous Cav1) and HT‐29 cells (which contain low endogenous Cav1) were transduced with a lentiviral construct expressing either Cav‐1 or Cav‐1 without a functional CSD (ΔCSD). HeLa cells were also genetically engineered to stably overexpress Cav‐1(Cav‐1 OE), ΔCSD, or enhanced green fluorescent protein (EGFP) as a comparative control. In all cases, migration was significantly greater in ΔCSD cells compared to Cav‐1 OE and control cells. Phosphorylated STAT‐3 was also decreased in Cav‐1 OE cells compared to control and ΔCSD cells. G2/M phase was significantly increased and G1 was decreased in ΔCSD cells compared to control and CAV1 OE cells. Cdc2 phosphorylation was also increased in ΔCSD cells. To further confirm that CSD affects cell migration, we performed migration assays in Hela cells expressing either a pEGFP tagged CSD or a scrambled sequence. CSD overexpressing cells showed a lower migration rate comparison to control cells. These findings suggest that Cav‐1 CSD affects cell migration, possibly by participating in a G2/M checkpoint. These findings may represent a new approach to the treatment of cancer metastasis.Support or Funding InformationNIH R01 HL091071This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

TGFβ1 Regulates KIM‐1 and Caveolin‐1 Expression through the Smad3 and ERK1/2 Signaling Pathways in Renal Cancer Cells

Kidney injury molecule‐1 (KIM‐1) is a type I transmembrane glycoprotein that is rapidly upregulated at the gene and protein level following various types of injury to the kidney. KIM‐1 expression is increased not only in acute and chronic kidney injury diseases but also in renal cell carcinoma. It may play an important role in carcinoma process. Caveolin‐1, the principal component of caveolae, is regarded as an important inhibitor of multiple signaling molecules. The cytokine transforming growth factor‐β1 (TGF‐β1) has been extensively studied in tumor biology and is believed to serve a variety of functions in tumor progression. In this study, we investigated the regulatory mechanism of TGF‐β1 on KIM‐1 and caveolin‐1 expression in 769‐P cells, a human renal cell adenocarcinoma cell line. Western blot analysis indicated that TGF‐β1 treatment resulted in a downregulation of KIM‐1 and an upregulation of caveolin‐1 in 769‐P cells. Specific knockdown of Smad3 with siRNAs significantly increased basal KIM‐1 expression and attenuated TGF‐β1‐induced KIM‐1 down regulation, which was associated with increased activation of extracellular signal‐regulated kinase (ERK) 1/2 and signal transducer and activator of transcription 3 (STAT3) signaling pathways. Interestingly, pre‐treatment of 769‐P cells with ERK inhibitor U0126 abolished TGF‐β1‐induced caveolin‐1 upregulation and partially restored KIM‐1 protein level. We also observed a significant decrease in KIM‐1 in 769‐P cells administered with caveolin‐1 scaffolding domain peptides. Taken together, our data demonstrate that TGF‐β1 may differentially regulate KIM‐1 and caveolin‐1 expression in renal cancer cells via both Smad3 and ERK1/2 signaling pathways.Support or Funding InformationNIH SC1DK112151; NIH/NCRR/RCMI 8G12MD007602 & 8U54MD007588This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Adipose-derived mesenchymal stromal/stem cells in systemic sclerosis: Alterations in function and beneficial effect on lung fibrosis are regulated by caveolin-1.

The potential value of mesenchymal stromal/stem cell therapy in treating skin fibrosis in scleroderma (systemic sclerosis) and of the caveolin-1 scaffolding domain peptide in treating lung, skin, and heart fibrosis is known. To understand how these observations may relate to differences between mesenchymal stromal/stem cells from healthy subjects and subjects with fibrosis, we have characterized the fibrogenic and adipogenic potential of adipose-derived mesenchymal stromal/stem cells from systemic sclerosis patients, from mice with fibrotic lung and skin disease induced by systemic bleomycin treatment, and from healthy controls. Early passage systemic sclerosis adipose-derived mesenchymal stromal/stem cells have a profibrotic/anti-adipogenic phenotype compared to healthy adipose-derived mesenchymal stromal/stem cells (low caveolin-1, high α-smooth muscle actin, high HSP47, low pAKT, low capacity for adipogenic differentiation). This phenotype is mimicked by treating healthy adipose-derived mesenchymal stromal/stem cells with transforming growth factor beta or caveolin-1 small interfering RNA and is reversed in systemic sclerosis adipose-derived mesenchymal stromal/stem cells by treatment with caveolin-1 scaffolding domain peptide, but not scrambled caveolin-1 scaffolding domain peptide. Similar results were obtained with adipose-derived mesenchymal stromal/stem cells from systemic sclerosis patients and from bleomycin-treated mice, indicating the central role of caveolin-1 in mesenchymal stromal/stem cell differentiation in fibrotic disease.

Downregulated Caveolin-1 expression in circulating monocytes may contribute to the pathogenesis of psoriasis

Caveolin-1 (CAV-1) is the principal component of caveolae that regulates a variety of signaling molecules and receptors. Our previous study revealed CAV-1 reduction in the epidermis of patients with psoriasis, which leads to enhanced Janus kinase/signal transducer and activator of transcription activation and cytokine production, suggesting that aberrant CAV-1 expression may contribute to psoriatic inflammation. This study aimed to investigate whether abnormal modulation of CAV-1 on immune cells is involved in the pathogenesis of psoriasis. We observed that CAV-1 level in psoriasis patients was apparently reduced in peripheral blood mononuclear cells (PBMCs) and it was prominent in CD14+ monocytes. CAV-1 silencing in monocytes represented elevated levels of interleukin (IL)-1β and IL-6, and those had enhanced chemotaxis activity. In a murine model of psoriasis-like inflammation induced by imiquimod, we observed a significant CAV-1 reduction in PBMCs. Systemic administration of CAV-1 scaffolding domain peptide significantly improved the skin phenotype with less macrophage infiltration. Taken together, aberrant CAV-1 expression in monocytes may be involved in the pathogenesis of psoriasis.

Suppression of angiotensin II-induced pathological changes in heart and kidney by the caveolin-1 scaffolding domain peptide.

Dysregulation of the renin-angiotensin system leads to systemic hypertension and maladaptive fibrosis in various organs. We showed recently that myocardial fibrosis and the loss of cardiac function in mice with transverse aortic constriction (TAC) could be averted by treatment with the caveolin-1 scaffolding domain (CSD) peptide. Here, we used angiotensin II (AngII) infusion (2.1 mg/kg/day for 2 wk) in mice as a second model to confirm and extend our observations on the beneficial effects of CSD on heart and kidney disease. AngII caused cardiac hypertrophy (increased heart weight to body weight ratio (HW/BW) and cardiomyocyte cross-sectional area); fibrosis in heart and kidney (increased levels of collagen I and heat shock protein-47 (HSP47)); and vascular leakage (increased levels of IgG in heart and kidney). Echocardiograms of AngII-infused mice showed increased left ventricular posterior wall thickness (pWTh) and isovolumic relaxation time (IVRT), and decreased ejection fraction (EF), stroke volume (SV), and cardiac output (CO). CSD treatment (i.p. injections, 50 μg/mouse/day) of AngII-infused mice significantly suppressed all of these pathological changes in fibrosis, hypertrophy, vascular leakage, and ventricular function. AngII infusion increased β1 and β3 integrin levels and activated Pyk2 in both heart and kidney. These changes were also suppressed by CSD. Finally, bone marrow cell (BMC) isolated from AngII-infused mice showed hyper-migration toward SDF1. When AngII-infused mice were treated with CSD, BMC migration was reduced to the basal level observed in cells from control mice. Importantly, CSD did not affect the AngII-induced increase in blood pressure (BP), indicating that the beneficial effects of CSD were not mediated via normalization of BP. These results strongly indicate that CSD suppresses AngII-induced pathological changes in mice, suggesting that CSD can be developed as a treatment for patients with hypertension and pressure overload-induced heart failure.

P53 Expression in Lung Fibroblasts Is Linked to Mitigation of Fibrotic Lung Remodeling

Idiopathic pulmonary fibrosis (IPF) is a debilitating, incurable, and life-threatening disease. A cardinal feature of the pathogenesis of IPF is excessive extracellular matrix deposition attributable to proliferation of activated fibrotic lung fibroblasts (fLfs). To assess the underlying mechanism, we analyzed the status of the tumor suppressor protein p53 in fLfs from the lungs of IPF patients or mice with bleomycin-induced established PF. We report that basal expression of p53 is markedly reduced in fLfs. Forced expression of caveolin-1 in fLfs increased basal p53 and reduced profibrogenic proteins, including collagen-1. Transduction of fLfs with adenovirus expressing p53 reduced expression of these proteins. Conversely, inhibition of baseline p53 in control lung fibroblasts from lung tissues increased profibrogenic protein expression. Lung transduction of adenovirus expressing p53 reduced bleomycin-induced PF in wild-type or caveolin-1-deficient mice. Furthermore, treatment of fLfs or fibrotic lung tissues with caveolin-1 scaffolding domain peptide (CSP) or its fragment, CSP7, restored p53 and reduced profibrogenic proteins. Treatment of wild-type mice with i.p. CSP or CSP7 resolved bleomycin-induced PF. These peptides failed to resolve PF in inducible conditional knockout mice lacking p53 in fLfs, indicating the induction of baseline fLf p53 as the basis of the antifibrotic effects.

Effect of caveolin-1 scaffolding domain peptides on heme oxygenase-1 activity increasing and M1/M2 phenotype polarization in rat alveolar macrophages induced by lipopolysaccharide

To investigate the effect of caveolin-1 scaffolding domain (CSD) peptides on heme oxygenase-1 (HO-1) activity increasing and M1/M2 phenotype polarization in rat alveolar macrophages (AMs) induced by lipopolysaccharide (LPS). Bioinformatics was used to analyze the binding of full-length wild-type CSD polypeptide and 101 amino acid deleted truncated mutant CSD polypeptide (Δ101CSD) to HO-1. Primary AMs were isolated from rats, when cell fusion reached 80%, they were synchronized with serum-free medium and divided into five groups: no treatment was given to the blank control group; LPS group was treated with 100 μg/L LPS for 16 hours; LPS + hemin group was treated with 100 μg/L LPS and 20 μmol/L hemin for 16 hours; wild-type CSD polypeptide + LPS + hemin group was pretreated with 10 μmol/L wild-type CSD polypeptide 6 hours before LPS treatment; Δ101CSD + LPS + hemin group was pretreated with 10 μmol/L Δ101CSD polypeptide 6 hours before LPS treatment. After treatment for 16 hours, the co-localization between caveolin-1 (Cav-1) and HO-1 was displayed by confocal microscope; the mRNA expressions of inflammatory cytokines interleukin-1β (IL-1β) and monocyte chemoattractant protein-1 (MCP-1) and M1/M2 polarization cytokines tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), leukocyte differentiation antigen 206 (CD206) and IL-10 were determined by real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-qPCR); the HO-1 activity and nitric oxide (NO) production were determined by spectrophotometry. Bioinformatics analysis showed: both wild-type CSD and Δ101CSD peptides could bind to HO-1, and there was no significant difference in the binding ability between the two peptides, but the deletion of 101 Arg resulted in the disappearance of part of the binding region between Δ101CSD and HO-1. The results of laser confocal microscopy showed: the expressions of Cav-1 and HO-1 were lowed in the blank control group, and Cav-1 was bound to HO-1 in LPS group and LPS + hemin group. Both wild-type CSD and Δ101CSD peptides pretreatment could significantly reduce the binding of HO-1 to Cav-1 induced by LPS. HO-1 activity analysis showed: after LPS stimulation, the activity of HO-1 was significantly higher than that of the blank control group; the activity of HO-1 induced by LPS was increased by hemin; after pretreatment with two kinds of CSD peptides, the activity of HO-1 was further increased, and the effect of wild-type CSD peptide was more significant, which showed a statistically significant difference as compared with that of LPS + hemin group (pmol×mg-1×h-1: 3 683±266 vs. 2 408±132, P < 0.05). RT-qPCR results showed: LPS could induce elevation of cytokines and M1 markers and decrease of M2 markers, while hemin could inhibit LPS-induced inflammatory response and M1/M2 phenotypic polarization. Compared with LPS + hemin group, after pretreatment with wild-type CSD peptide, the levels of inflammatory factors in AMs were decreased, and the mRNA expression levels of TNF-α and iNOS, M1 markers, were decreased [TNF-α mRNA (2-ΔΔCt): 6.82±0.05 vs. 8.70±0.24, iNOS mRNA (2-ΔΔCt): 331.50±32.05 vs. 506.70±0.10, both P < 0.05], and IL-10 mRNA expression level was increased (2-ΔΔCt: 269.09±6.54 vs. 119.05±3.30, P < 0.05). The deletion of 101 site partially weakened the inhibitory effect of CSD peptides on inflammatory factors and only reduced the expression of iNOS mRNA (2-ΔΔCt: 429.11±8.92 vs. 506.70±0.10, P < 0.05), indicating that its ability to transform AMs from M1 phenotype to M2 phenotype was poor. The two peptides had no effect on the expression of CD206. Wild-type CSD had beneficial effects of anti-inflammation by reducing Cav-1 binding to HO-1 induced by LPS, restoring the HO-1 activity and driving M2 phenotype in alveolar macrophages.

Deletion 101 residue at caveolin-1 scaffolding domain peptides impairs the ability of increasing heme oxygenase-1 activity

ObjectiveResident alveolar macrophages (AMs) are activated and release proinflammatory mediators and chemokines during acute lung injury. We have previous reported that caveolin-1 (Cav-1) scaffolding domain (CSD) peptide inhibited the proinflammatory cytokines expression by up-regulating heme oxygenase-1 (HO-1) activity. In this study, we aimed to investigate the effect of residue R101 in CSD peptide on the activity of HO-1 in AMs. MethodsThe binding mode between HO-1 and CSD peptides (WT CSD and Δ101 CSD truncation peptides) was analyzed and the free energy was calculated. The inflammatory genes and M1/M2macrophage polarization-associated genes expression were measured by real-time PCR. The activities of HO-1 were determined by the spectrophotometical method. Western blot analyzed the content of Cav-1, HO-1, IκB and MAPK signals (phosphorylated ERK, JNK and p38 MAPK). ResultsΔ101CSD peptide could bind to HO-1 protein and to disrupt the interaction of HO-1 and Cav-1. However, Δ101CSD peptide had lower activity of HO-1 in LPS-treated AMs compared with WT CSD. The expression of IL-1β and MCP-1 and NO content were decreased by WT CSD peptide in LPS treated AMs. However, only MCP-1 expression and NO content were downregulated byΔ101CSD peptide. Meanwhile, compared with those in LPS + hemin + WT CSD group, the mRNA expression of TNF-α, Cd86, IL-12b and NOS2 significantly increased while expression of IL10, Arg1 and CD163 significantly decreased in LPS + hemin + Δ101CSD group. The effect of WT CSD peptide on the inhibition of MAPK signaling pathway were stronger than Δ101 CSD peptide evidenced by the level of phosphorylated ERK, JNK and p38 MAPK. ConclusionDeletion of residue R101 impairs the ability of CSD peptide to increase HO-1 activity and to dampen inflammatory response in LPS-challenged rat AMs.

Caveolin-1 Scaffolding Domain Peptides Alleviate Liver Fibrosis by Inhibiting TGF-β1/Smad Signaling in Mice.

Liver fibrosis is the common pathological process characterized by activation of hepatic stellate cells (HSCs) and overproduction of extracellular matrix (ECM). Caveolin-1 (Cav1), the principal component of caveolae, is regarded as an important inhibitor of multiple signaling molecules including transforming growth factor β1(TGF-β1) signaling. To evaluate the role of Cav1 in liver fibrosis, Cav1 deficient (Cav1−/−) and wild type (WT) mice were subjected to liver fibrosis induced by carbon tetrachloride (CCl4). Results indicated no significant difference between Cav1−/− and WT mice in inflammation or collagen content before CCl4 treatment. After CCl4 administration, Cav1−/− mice showed enhanced TGF-β1 signaling, as reflected by a significantly greater amount of phosphorylation of Smad2 and collagen deposition in livers over WT animals. Qualitative and quantitative analysis indicated that inflammatory injury to the liver was markedly aggravated, accompanied by increased degeneration and necrosis of hepatocytes, higher alanine aminotransferase (ALT)/aspartate aminotransferase (AST), TGF-α and IL-1β levels in Cav1−/− animals. The mRNA and protein levels of α-smooth muscle actin (α-SMA), Collagen α1(I), and Collagen α1(III) were further enhanced in Cav1−/− animals. We also observed a significant decrease in collagen content in Cav1−/− and WT animals administrated with Cav1 scaffolding domain peptides (CSD). In vitro study indicated that phosphorylation of Smad2 was inhibited after CSD treatment, accompanied by decreased protein levels of α-SMA, Collagen α1(I), and Collagen α1(III) in HSCs. We conclude that Cav1 is an important inhibitor of TGF-β1/Smad signaling in HSCs activation and collagen production, which might make it a promising target for therapy of liver fibrosis.

Reduction of Endothelial Nitric Oxide Increases the Adhesiveness of Constitutive Endothelial Membrane ICAM-1 through Src-Mediated Phosphorylation.

Nitric oxide (NO) is a known anti-adhesive molecule that prevents platelet aggregation and leukocyte adhesion to endothelial cells (ECs). The mechanism has been attributed to its role in the regulation of adhesion molecules on leukocytes and the adhesive properties of platelets. Our previous study conducted in rat venules found that reduction of EC basal NO synthesis caused EC ICAM-1-mediated firm adhesion of leukocytes within 10–30 min. This quick response occurred in the absence of alterations of adhesion molecules on leukocytes and also opposes the classical pattern of ICAM-1-mediated leukocyte adhesion that requires protein synthesis and occurs hours after stimulation. The objective of this study is to investigate the underlying mechanisms of reduced basal NO-induced EC-mediated rapid leukocyte adhesion observed in intact microvessels. The relative levels of ICAM-1 at different cell regions and their activation status were determined with cellular fractionation and western blot using cultured human umbilical vein ECs. ICAM-1 adhesiveness was determined by immunoprecipitation in non-denatured proteins to assess the changes in ICAM-1 binding to its inhibitory antibody, mAb1A29, and antibody against total ICAM-1 with and without NO reduction. The adhesion strength of EC ICAM-1 was assessed by atomic force microscopy (AFM) on live cells. Results showed that reduction of EC basal NO caused by the application of caveolin-1 scaffolding domain (AP-CAV) or NOS inhibitor, L-NMMA, for 30 min significantly increased phosphorylated ICAM-1 and its binding to mAb1A29 in the absence of altered ICAM-1 expression and its distribution at subcellular regions. The Src inhibitor, PP1, inhibited NO reduction-induced increases in ICAM-1 phosphorylation and adhesive binding. AFM detected significant increases in the binding force between AP-CAV-treated ECs and mAb1A29-coated probes. These results demonstrated that reduced EC basal NO lead to a rapid increase in ICAM-1 adhesive binding via Src-mediated phosphorylation without de novo protein synthesis and translocation. This study suggests that a NO-dependent conformational change of constitutive EC membrane ICAM-1 might be the mechanism of rapid ICAM-1 dependent leukocyte adhesion observed in vivo. This new mechanistic insight provides a better understanding of EC/leukocyte interaction-mediated vascular inflammation under many disease conditions that encounter reduced basal NO in the circulation system.

The PhosphooCaveolinn1 Scaffolding Domain Dampens Force Fluctuations in Focal Adhesions to Drive Cancer Cell Migration

Caveolin‐1 (Cav1), a major Src kinase substrate phosphorylated on tyrosine‐14 (Y14), contains the highly‐conserved membrane‐proximal caveolin scaffolding domain (CSD; amino acids 82‐101). Here we show, using CSD mutants (F92A/V94A) and membrane‐permeable CSD competing peptides, that Src kinase‐dependent pY14Cav1 regulation of focal adhesion protein stabilization, focal adhesion tension and cancer cell migration is CSD‐dependent. Quantitative proteomic analysis of Cav1‐GST (1‐101 amino acids) pulldowns showed 6‐fold increased binding of vinculin and, to a lesser extent, α‐actinin, talin and filamin, to phosphomimetic Cav1Y14D relative to non‐phosphorylatable Cav1Y14F. Consistently, pY14Cav1 enhanced CSD‐dependent vinculin tension in focal adhesions, dampening force fluctuation and synchronously stabilizing cellular focal adhesions in a high‐tension mode, paralleling effects of actin stabilization. By promoting focal adhesion traction, functional interaction between Cav1 Y14 phosphorylation and the CSD shifts the molecular clutch linking adhesions and the actin cytoskeleton to high gear, and thereby drives cancer cell migration.