Grzegorz Sowa

Here we report that membrane protein caveolin‐2 (Cav‐2) is regulated during activation of primary mouse peritoneal macrophages and RAW264.7 macrophage cell line by a bacterial lipopolysaccharide (LPS) and a cytokine, interferon gamma (IFN‐γ). We also show that downregulation or loss of Cav‐2 increases expression of inducible nitric oxide (NO) synthase (iNOS) and subsequent NO production in activated macrophages. Treatment with LPS and IFN‐γ downregulated Cav‐2 at the protein and mRNA level. Moreover, LPS‐ and IFN‐γ‐induced downregulation of Cav‐2 inversely correlated with iNOS expression levels. Mechanistically, activation of NF‐κB pathway was responsible for downregulation of Cav‐2 by LPS and IFN‐γ since pharmacological inhibition of NF‐κB activation with pyrrolidine dithiocarbamate (PDTC) prevented Cav‐2 downregulation and iNOS activation. To test if LPS‐ and IFN‐γ‐induced downregulation of Cav‐2 could possibly be involved in macrophage activation, we used a combination of siRNA knockdown and genetic deletion/knockout (KO) approaches. Remarkably, reduction of Cav‐2 using siRNA approach resulted in enhanced STAT1 phosphorylation, iNOS expression, and increased NO production in LPS‐ and LPS plus IFN‐γ‐stimulated macrophages. Conversely, genetic deletion of Cav‐2 robustly enhanced IFN‐γ‐stimulated iNOS expression and subsequent NO production. Overall, our data suggest that Cav‐2 is not only regulated during macrophage activation, but it also may be an important physiological regulator of macrophage activation via preventing excessive STAT1 signaling and iNOS stimulation.

The role of caveolin-2 in regulating ischemia/reperfusion (I/R) tissue injury and the mechanisms underlying its effects are unknown. This study uses caveolin-2-deficient mouse and small intestinal I/R injury models to examine the role of caveolin-2 in the leukocyte-dependent reperfusion injury. We demonstrate for the first time that caveolin-2 plays a protective role from the I/R-induced leukocyte-dependent reperfusion injury by reducing PAI-1 protein levels in intestinal tissue and leukocyte-endothelial adhesive interactions in postcapillary venules.

Immunosuppression is critical for tumor growth and metastasis as well as obstacle to effective immunotherapy. Here, we demonstrate that host deficiency in caveolin-2, a member of caveolin protein family, increases M1-polarized tumor-associated macrophage (TAM) and CD8 T cell infiltration into subcutaneously implanted murine lung carcinoma tumors. Importantly, increase in M1 TAM-specific markers and cytokines occurs prior to increased numbers of tumor-infiltrating CD8 T cells and tumor regression in caveolin-2 deficient mice, suggesting that an early increase in M1 TAMs is a novel mechanism, via which host deficiency in caveolin-2 inhibits tumor growth. Consistent with the latter, transfer and co-injection of caveolin-2 deficient bone marrow (origin of TAMs) suppresses tumor growth and increases numbers of M1-polarized TAMs in wild type mice. Collectively, our data suggest that lung cancer cells use caveolin-2 expressed in bone marrow-derived cell types including TAMs to promote tumor growth via suppressing the anti-tumor immune response and that caveolin-2 could be a potential target for cancer immunotherapy.

Caveolin-2 (Cav2) is a major protein component of caveolae in membranes of vascular smooth muscle and endothelium, yet its absence alters the ultrastructure of skeletal muscle fibers. To gain insight into Cav2 function in skeletal muscle, we tested the hypothesis that genetic deletion of Cav2 would alter microvascular reactivity and depress contractile function of skeletal muscle in vivo. In the left gluteus maximus muscle (GM) of anesthetized Cav2^−/− and wild-type (WT) male mice (age, 6 mo), microvascular responses to physiological agonists and to GM contractions were studied at 34°C. For feed arteries (FA), first- (1A), second- (2A) and third-order (3A) arterioles, respective mean diameters at rest (45, 35, 25, 12 μm) and during maximal dilation (65, 55, 45, 30 μm) were similar between groups. Cumulative dilations to ACh (10⁻⁹ to 10⁻⁵ M) and constrictions to norepinephrine (10⁻⁹ to 10⁻⁵ M) were also similar between groups, as were steady-state dilations during rhythmic twitch contractions (2 and 4 Hz; 30 s). For single tetanic contractions (100 Hz; 100, 250, and 500 ms), rapid onset vasodilation (ROV) increased with contraction duration throughout networks in GM of both groups but was reduced by nearly half in Cav2^−/− mice compared with WT mice ( P < 0.05). Nevertheless, maximal force during tetanic contraction was ∼40% greater in GM of Cav2^−/− vs. WT mice (152 ± 14 vs. 110 ± 3 mN per square millimeter, respectively; P < 0.05). Thus, while structural and functional properties of resistance networks are well maintained in the GM of Cav2^−/− mice, diminished ROV with greater force production reveals novel physiological roles for Cav2 in skeletal muscle.

In addition to cancer cells, primary tumors are composed of a multitude of stromal cell types. Among others, the stromal cell types involved in tumor growth and progression include endothelial cells, fibroblasts, pericytes, stem cells and various cell types of immune origin. While the role of oncogenes or tumor suppressor proteins expressed in cancer cells has been extensively studied, far less is known about potential involvement of proteins expressed in stromal cell types present within the tumor microenvironment. Recent experimental evidence from our laboratory suggests that caveolin-2 (Cav-2) protein expressed in stromal cell types of the tumor microenvironment promotes subcutaneous tumor growth in two independent syngeneic mouse models, i.e., Lewis lung carcinoma (LLC) and B16-F10 melanoma. Mechanistically, the tumor growth promoting role of Cav-2 is associated with enhanced tumor induced neovascularization. At the molecular level, host-expressed Cav-2 appears to prevent excessive expression of anti-angiogenic thrombospondin-1 (TSP-1) and promote phosphorylation of pro-angiogenic endothelial nitric oxide synthase (eNOS) at serine 1177. Taken together, our recent findings suggest that Cav-2 expressed within the tumor microenvironment could be a potential target for anti-cancer therapy.

Caveolin-2 (Cav-2), a member of caveolin protein family, is largely different from better known caveolin-1 (Cav-1) and thus might play distinct functions. Here, we provide the first genetic evidence suggesting that host-expressed Cav-2 promotes subcutaneous tumor growth and tumor-induced neovascularization using two independent syngeneic mouse models. Host deficiency in Cav-2 resulted in defective and reduced growth of subcutaneously implanted Lewis lung carcinoma (LLC) and B16-F10 melanoma tumors, respectively. Consistent with the defective growth, LLC and B16-F10 melanoma tumors implanted into Cav-2 KO mice displayed reduced microvascular density (MVD) determined by IHC with anti-CD31 antibodies, suggesting impaired pathologic angiogenesis. Additional studies involving LLC tumors extracted from Cav-2 KO mice just 10 days after implantation determined reduced cell proliferation, massive necrotic cell death, and fibrosis. In contrast with day 10, only MVD but not cell proliferation and survival was reduced in the earliest palpable LLC tumors extracted 6 days after implantation into Cav-2 KO mice, suggesting that impaired angiogenesis is the causative factor. Mechanistically, impaired LLC tumor growth and angiogenesis in Cav-2 KO mice was associated with increased expression levels of antiangiogenic thrombospondin-1 and inhibited S1177 phosphorylation of endothelial nitric oxide synthase. Taken together, our data suggest that host deficiency in Cav-2 impairs tumor-induced angiogenesis, leading to compromised tumor cell survival/proliferation manifested by the defective tumor growth. In conclusion, host-expressed Cav-2 may promote tumor growth via supporting tumor-induced angiogenesis. Thus, Cav-2 expressed in tumor microenvironment may potentially become a novel target for cancer therapy. Cancer Res; 74(22); 6452–62. ©2014 AACR.

Here we show that tyrosine phosphorylation of caveolin‐2 (Cav‐2) negatively regulates the anti‐proliferative function of transforming growth factor beta (TGF‐beta) in endothelial cells. In contrast to wild‐type‐Cav‐2, retroviral re‐expression of Y19/27F‐Cav‐2 in Cav‐2 knockout endothelial cells did not affect anti‐proliferative effect of TGF‐beta compared to empty vector. Conversely, although less effective than wild‐type, re‐expression of S23/36A‐Cav‐2 reduced the effect of TGF‐beta compared to empty vector. This differential effect of tyrosine and serine phosphorylation mutants of Cav‐2 correlated with TGF‐beta‐induced Smad3 phosphorylation and transcriptional activation of plasminogen activator inhibitor‐1. Thus tyrosine‐phosphorylated Cav‐2 counteracts anti‐proliferative effect of TGF‐beta in endothelial cells.

Caveolae are cholesterol and glycosphingolipid-rich flask-shaped invaginations of the plasma membrane which are particularly abundant in vascular endothelium and present in all other cell types of the cardiovascular system, including vascular smooth-muscle cells, macrophages, cardiac myocytes, and fibroblasts. Caveolins and the more recently discovered cavins are the major protein components of caveolae. When caveolae were discovered, their functional role was believed to be limited to transport across the endothelial cell barrier. Since then, however, a large body of evidence has accumulated, suggesting that these microdomains are very important in regulating many other important endothelial cell functions, mostly due to their ability to concentrate and compartmentalize various signaling molecules. Over the course of several years, multiple studies involving knockout mouse and small interfering RNA approaches have considerably enhanced our understanding of the role of caveolae and caveolin-1 in regulating many cardiovascular functions. New findings have been reported implicating other caveolar protein components in endothelial cell signaling and function, such as the understudied caveolin-2 and newly discovered cavin proteins. The aim of this review is to focus primarily on molecular and cellular aspects of the role of caveolae, caveolins, and cavins in endothelial cell signaling and function. In addition, where appropriate, the possible implications for the cardiovascular and pulmonary physiology and pathophysiology will be discussed.

Caveolin-2 is one of the major protein components of cholesterol- and glycosphingolipid-rich flask-shaped invaginations of plasma membrane caveolae. A new body of evidence suggests that caveolin-2 plays an important, and often more direct, role than caveolin-1 in regulating signaling and function in a cell- and tissue type-specific manner. The purpose of this paper is to primarily focus on discussing how these recent discoveries may help better understand the specific contribution of caveolin-2 to lipid raft- and caveolae-regulated cell/tissue-specific signaling and functions.

Using a combination of wild-type (WT) and caveolin-2 (Cav-2) knockout along with retroviral reexpression approaches, we provide the evidence for the negative role of Cav-2 in regulating anti-proliferative function and signaling of transforming growth factor β (TGF-β) in endothelial cells (ECs). Although, TGF-β had a modest inhibitory effect on WT ECs, it profoundly inhibited proliferation of Cav-2 knockout ECs. To confirm the specificity of the observed difference in response to TGF-β, we have stably reexpressed Cav-2 in Cav-2 knockout ECs using a retroviral approach. Similar to WT ECs, the anti-proliferative effect of TGF-β was dramatically reduced in the Cav-2 reexpressing ECs. The reduced anti-proliferative effect of TGF-β in Cav-2-positive cells was evidenced by three independent proliferation assays: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cell count, and bromodeoxyuridine incorporation and correlated with a loss of TGF-β-mediated upregulation of cell cycle inhibitor p27 and subsequent reduction of the levels of hyperphosphorylated (inactive) form of the retinoblastoma protein in Cav-2 reexpressing ECs. Mechanistically, Cav-2 inhibits anti-proliferative action of TGF-β by suppressing Alk5-Smad2/3 pathway manifested by reduced magnitude and length of TGF-β-induced Smad2/3 phosphorylation as well as activation of activin receptor-like kinase-5 (Alk5)-Smad2/3 target genes plasminogen activator inhibitor-1 and collagen type I in Cav-2-positive ECs. Expression of Cav-2 does not appear to significantly change targeting of TGF-β receptors I and Smad2/3 to caveolar and lipid raft microdomains as determined by sucrose fractionation gradient. Overall, the negative regulation of TGF-β signaling and function by Cav-2 is independent of Cav-1 expression levels and is not because of changing targeting of Cav-1 protein to plasma membrane lipid raft/caveolar domains.