Alex W. Cohen

Caveolin-1/3 Double-Knockout Mice Are Viable, but Lack Both Muscle and Non-Muscle Caveolae, and Develop a Severe Cardiomyopathic Phenotype

The caveolin gene family consists of caveolins 1, 2, and 3. Caveolins 1 and 2 are co-expressed in many cell types, such as endothelial cells, fibroblasts, smooth muscle cells and adipocytes, where they form a heteroligomeric complex. In contrast, the expression of caveolin-3 is muscle-specific. Thus, the expression of caveolin-1 is required for caveolae formation in non-muscle cells, while the expression of caveolin-3 drives caveolae formation in striated muscle cell types (cardiac and skeletal). To create a truly caveolae-deficient mouse, we interbred Cav-1 null mice and Cav-3 null mice to generate Cav-1/Cav-3 double-knockout (Cav-1/3 dKO) mice. Here, we report that Cav-1/3 dKO mice are viable and fertile, despite the fact that they lack morphologically identifiable caveolae in endothelia, adipocytes, smooth muscle cells, skeletal muscle fibers, and cardiac myocytes. We also show that these mice are deficient in all three caveolin gene products, as caveolin-2 is unstable in the absence of caveolin-1. Interestingly, Cav-1/3 dKO mice develop a severe cardiomyopathy. At 2 months of age, analysis of Cav-1/3 dKO hearts via gated magnetic resonance imaging reveals a dramatic increase in left ventricular wall thickness, as compared with Cav-1-KO, Cav-3 KO, and wild-type mice. Further functional analysis of Cav-1/3 dKO hearts via transthoracic echocardiography demonstrates hypertrophy and dilation of the left ventricle, with a significant decrease in fractional shortening. As predicted, Northern analysis of RNA derived from the left ventricle of Cav-1/3 dKO mice shows a dramatic up-regulation of the atrial natriuretic factor message, a well-established biochemical marker of cardiac hypertrophy. Finally, histological analysis of Cav-1/3 dKO hearts reveals hypertrophy, disorganization, and degeneration of the cardiac myocytes, as well as chronic interstitial fibrosis and inflammation. Thus, dual ablation of both Cav-1 and Cav-3 genes in mice leads to a pleiotropic defect in caveolae formation and severe cardiomyopathy.

Caveolin-1-Deficient Mice Show Defects in Innate Immunity and Inflammatory Immune Response during Salmonella enterica Serovar Typhimurium Infection†

ABSTRACT A number of studies have shown an association of pathogens with caveolae. To this date, however, there are no studies showing a role for caveolin-1 in modulating immune responses against pathogens. Interestingly, expression of caveolin-1 has been shown to occur in a regulated manner in immune cells in response to lipopolysaccharide (LPS). Here, we sought to determine the role of caveolin-1 (Cav-1) expression in Salmonella pathogenesis. Cav-1−/− mice displayed a significant decrease in survival when challenged with Salmonella enterica serovar Typhimurium. Spleen and tissue burdens were significantly higher in Cav-1−/− mice. However, infection of Cav-1−/− macrophages with serovar Typhimurium did not result in differences in bacterial invasion. In addition, Cav-1−/− mice displayed increased production of inflammatory cytokines, chemokines, and nitric oxide. Regardless of this, Cav-1−/− mice were unable to control the systemic infection of Salmonella. The increased chemokine production in Cav-1−/− mice resulted in greater infiltration of neutrophils into granulomas but did not alter the number of granulomas present. This was accompanied by increased necrosis in the liver. However, Cav-1−/− macrophages displayed increased inflammatory responses and increased nitric oxide production in vitro in response to Salmonella LPS. These results show that caveolin-1 plays a key role in regulating anti-inflammatory responses in macrophages. Taken together, these data suggest that the increased production of toxic mediators from macrophages lacking caveolin-1 is likely to be responsible for the marked susceptibility of caveolin-1-deficient mice to S. enterica serovar Typhimurium.

Caveolin-1-Deficient Mice Have An Increased Mammary Stem Cell Population with Upregulation of Wnt/?-Catenin Signaling

Here, we show that a caveolin-1 (Cav-1) deficiency leads to an amplification of the adult mammary stem cell population, both in vivo and in vitro. First, the expression of two stem cell markers, Sca-1 and Keratin 6, is dramatically increased in the hyperplastic mammary ducts of Cav-1 deficient mice, suggesting that loss of Cav-1 induces the accumulation of a progenitor cell population in the mammary gland. To independently validate these results, we reconstituted mammary acini formation in vitro via a 3D Matrigel assay system--using primary cultures of mammary epithelial cells derived from WT and Cav-1 deficient mice. We show that Cav-1 null 3D epithelial structures display an intense increase in the expression of three stem cell markers, i.e., Sca-1, keratin 6 and keratin 5. Overall, we observed a 2-to-3 fold increase in the number of Cav-1 KO acini that are positive for a given stem cell marker. Also, we show that such amplification of progenitor cells has functional consequences, as demonstrated by the abnormal presence of myoepithelial cells in the hyperplastic lesions of Cav-1 deficient mammary glands. Finally, we provide evidence that hyper-activation of Wnt/?-catenin signaling may constitute one of the down-stream mechanisms leading to mammary stem cell accumulation. The longevity and slow-dividing properties of mammary stem cells facilitates the accumulation of genetic alterations, and renders these progenitor cells the likely precursors of malignant derivatives. As such, we propose that loss of Cav-1 induces the accumulation of mammary stem cells, and that this event may be an initiating factor during mammary tumorigenesis.

Segregation of micron-scale membrane sub-domains in live murine sperm.

Lipid rafts, membrane sub‐domains enriched in sterols and sphingolipids, are controversial because demonstrations of rafts have often utilized fixed cells. We showed in living sperm that the ganglioside GM1 localized to a micron‐scale membrane sub‐domain in the plasma membrane overlying the acrosome. We investigated four models proposed for membrane sub‐domain maintenance. GM1 segregation was maintained in live sperm incubated under non‐capacitating conditions, and after sterol efflux, a membrane alteration necessary for capacitation. The complete lack of GM1 diffusion to the post‐acrosomal plasma membrane (PAPM) in live cells argued against the transient confinement zone model. However, within seconds after cessation of sperm motility, GM1 dramatically redistributed several microns from the acrosomal sub‐domain to the post‐acrosomal, non‐raft sub‐domain. This redistribution was not accompanied by movement of sterols, and was induced by the pentameric cholera toxin subunit B (CTB). These data argued against a lipid–lipid interaction model for sub‐domain maintenance. Although impossible to rule out a lipid shell model definitively, mice lacking caveolin‐1 maintained segregation of both sterols and GM1, arguing against a role for lipid shells surrounding caveolin‐1 in sub‐domain maintenance. Scanning electron microscopy of sperm freeze‐dried without fixation identified cytoskeletal structures at the sub‐domain boundary. Although drugs used to disrupt actin and intermediate filaments had no effect on the segregation of GM1, we found that disulfide‐bonded proteins played a significant role in sub‐domain segregation. Together, these data provide an example of membrane sub‐domains extreme in terms of size and stability of lipid segregation, and implicate a protein‐based membrane compartmentation mechanism.

Activation of Transcription Factors AP-1 and NF-κB in Murine Chagasic Myocarditis

ABSTRACT The myocardium of CD1 mice was examined for the activation of signal transduction pathways leading to cardiac inflammation and subsequent remodeling during Trypanosoma cruzi infection (Brazil strain). The activity of three pathways of the mitogen-activated protein kinases (MAPKs) was determined. Immunoblotting revealed a persistent elevation of phosphorylated (activated) extracellular-signal-regulated kinase (ERK), which regulates cell proliferation. During infection there was a transient activation of p38 MAPK but no activation of Jun N-terminal kinase. Early targets of activated ERK, c-Jun and c-Fos, were elevated during infection, as demonstrated by semiquantitative reverse transcription-PCR. Immunostaining revealed that the endothelium and the interstitial cells were most intensely stained with antibodies to c-Jun and c-Fos. Soon after infection, AP-1 and NF-κB DNA binding activity was increased. Protein levels of cyclin D1, the downstream target of ERK and NF-κB, were induced during acute infection. Immunostaining demonstrated increased expression of cyclin D1 in the vascular and endocardial endothelium, inflammatory cells, and the interstitial areas. Increased expression of the cyclin D1-specific phosphorylated retinoblastoma protein (Ser780) was also evident. Immunoblotting and immunostaining also demonstrated increased expression of proliferating cellular nuclear antigen that was predominantly present in the inflammatory cells, interstitial areas (i.e., fibroblasts), and endothelium. These data demonstrate that T. cruzi infection results in activation of the ERK-AP-1 pathway and NF-κB. Cyclin D1 expression was also increased. These observations provide a molecular basis for the activation of pathways involved in cardiac remodeling in chagasic cardiomyopathy.

Activation of transcription factors AP-1 and NF-kappa B in murine Chagasic myocarditis.

ABSTRACT The myocardium of CD1 mice was examined for the activation of signal transduction pathways leading to cardiac inflammation and subsequent remodeling during Trypanosoma cruzi infection (Brazil strain). The activity of three pathways of the mitogen-activated protein kinases (MAPKs) was determined. Immunoblotting revealed a persistent elevation of phosphorylated (activated) extracellular-signal-regulated kinase (ERK), which regulates cell proliferation. During infection there was a transient activation of p38 MAPK but no activation of Jun N-terminal kinase. Early targets of activated ERK, c-Jun and c-Fos, were elevated during infection, as demonstrated by semiquantitative reverse transcription-PCR. Immunostaining revealed that the endothelium and the interstitial cells were most intensely stained with antibodies to c-Jun and c-Fos. Soon after infection, AP-1 and NF-κB DNA binding activity was increased. Protein levels of cyclin D1, the downstream target of ERK and NF-κB, were induced during acute infection. Immunostaining demonstrated increased expression of cyclin D1 in the vascular and endocardial endothelium, inflammatory cells, and the interstitial areas. Increased expression of the cyclin D1-specific phosphorylated retinoblastoma protein (Ser780) was also evident. Immunoblotting and immunostaining also demonstrated increased expression of proliferating cellular nuclear antigen that was predominantly present in the inflammatory cells, interstitial areas (i.e., fibroblasts), and endothelium. These data demonstrate that T. cruzi infection results in activation of the ERK-AP-1 pathway and NF-κB. Cyclin D1 expression was also increased. These observations provide a molecular basis for the activation of pathways involved in cardiac remodeling in chagasic cardiomyopathy.

Cell Cycle Regulatory Proteins in the Liver in Murine Trypanosoma cruzi Infection

The liver is an important target of Trypanosoma cruzi infection. Infection of CD-1 mice withT. cruzi (Brazil strain) resulted in parasitism of the liver, primarily in sinusoidal and Kupffercells. Immunoblot analysis revealed activation of extra cellular signal-regulated kinase (ERK)during the acute and subacute period of infection, but p38 mitogen activated kinase (MAPK) andJNK were not activated. The activity of important cell cycle regulatory genes was also examinedin the liver following infection. There was increased expression of cyclin D1, cyclin E and cyclinA as well as proliferating cell nuclear antigen (PCNA) at 45, 60 and 215 days post infection. Inaddition, the expression of the cyclin-dependent kinase inhibitors p27KIP1, p21WAF1 and the tumorsuppressor p53 were increased in the liver obtained from infected mice. Quantitative PCRrevealed increased abundance of mRNA for cyclins A, D1 and E. Interestingly, cyclin A and Eare ordinarily not found in the adult liver. Thus infection caused a reversion to a fetal/neonatalphenotype. These data provide a molecular basis for cell proliferation in the liver following T.cruzi infection.

Activation of Transcription Factors AP-1 and NF- B in Murine Chagasic Myocarditis

ABSTRACT The myocardium of CD1 mice was examined for the activation of signal transduction pathways leading to cardiac inflammation and subsequent remodeling during Trypanosoma cruzi infection (Brazil strain). The activity of three pathways of the mitogen-activated protein kinases (MAPKs) was determined. Immunoblotting revealed a persistent elevation of phosphorylated (activated) extracellular-signal-regulated kinase (ERK), which regulates cell proliferation. During infection there was a transient activation of p38 MAPK but no activation of Jun N-terminal kinase. Early targets of activated ERK, c-Jun and c-Fos, were elevated during infection, as demonstrated by semiquantitative reverse transcription-PCR. Immunostaining revealed that the endothelium and the interstitial cells were most intensely stained with antibodies to c-Jun and c-Fos. Soon after infection, AP-1 and NF-κB DNA binding activity was increased. Protein levels of cyclin D1, the downstream target of ERK and NF-κB, were induced during acute infection. Immunostaining demonstrated increased expression of cyclin D1 in the vascular and endocardial endothelium, inflammatory cells, and the interstitial areas. Increased expression of the cyclin D1-specific phosphorylated retinoblastoma protein (Ser780) was also evident. Immunoblotting and immunostaining also demonstrated increased expression of proliferating cellular nuclear antigen that was predominantly present in the inflammatory cells, interstitial areas (i.e., fibroblasts), and endothelium. These data demonstrate that T. cruzi infection results in activation of the ERK-AP-1 pathway and NF-κB. Cyclin D1 expression was also increased. These observations provide a molecular basis for the activation of pathways involved in cardiac remodeling in chagasic cardiomyopathy.

Caveolin-1 Promotes Tumor Progression in an Autochthonous Mouse Model of Prostate Cancer: Genetic Ablation of Cav-1 Delays Advanced Prostate Tumor Development in Tramp Mice

Caveolin-1 (Cav-1) is the primary structural component of caveolae and is implicated in the processes of vesicular transport, cholesterol balance, transformation, and tumorigenesis. Despite an abundance of data suggesting that Cav-1 has transformation suppressor properties both in vitro and in vivo, Cav-1 is expressed at increased levels in human prostate cancer. To investigate the role of Cav-1 in prostate cancer onset and progression, we interbred Cav-1(-/-) null mice with a TRAMP (transgenic adenocarcinoma of mouse prostate) model that spontaneously develops advanced prostate cancer and metastatic disease. We found that, although the loss of Cav-1 did not affect the appearance of minimally invasive prostate cancer, its absence significantly impeded progression to highly invasive and metastatic disease. Inactivation of one (+/-) or both (-/-) alleles of Cav-1 resulted in significant reductions in prostate tumor burden, as well as decreases in regional lymph node metastases. Moreover, further examination revealed decreased metastasis to distant organs, such as the lungs, in TRAMP/Cav-1(-/-) mice. Utilizing prostate carcinoma cell lines (C1, C2, and C3) derived from TRAMP tumors, we also showed a positive correlation between Cav-1 expression and the ability of these cells to form tumors in vivo. Furthermore, down-regulation of Cav-1 expression in these cells, using a small interfering RNA approach, significantly reduced their tumorigenic and metastatic potential. Mechanistically, we showed that loss or down-regulation of Cav-1 expression results in increased apoptosis, with increased prostate apoptosis response factor-4 and PTEN levels in Cav-1(-/-) null prostate tumors. Our current findings provide the first in vivo molecular genetic evidence that Cav-1 does indeed function as a tumor promoter during prostate carcinogenesis, rather than as a tumor suppressor.