The molecular and cellular basis of coronavirus neurovirulence is poorly understood. Since neurovirulence may be determined at the early stages of infection of the central nervous system (CNS), we hypothesize that it may depend on the ability of the virus to induce proinflammatory signals from brain cells for the recruitment of blood-derived inflammatory cells. To test this hypothesis, we studied the interaction between coronaviruses (mouse hepatitis virus) of different neurovirulences with primary cell cultures of brain immune cells (astrocytes and microglia) and mouse tissues. We found that the level of neurovirulence of the virus correlates with its differential ability to induce proinflammatory cytokines (interleukin 12 [IL-12] p40, tumor necrosis factor alpha, IL-6, IL-15, and IL-1␤) in astrocytes and microglia and in mouse brains and spinal cords. These findings suggest that coronavirus neurovirulence may depend on a novel discriminatory ability of astrocytes and microglia to induce a proinflammatory response in the CNS.

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Increasing chronological age is the most significant risk factor for human cancer development. To examine the effects of host aging on mammary tumor growth, we used caveolin (Cav)-1 knockout mice as a bona fide model of accelerated host aging. Mammary tumor cells were orthotopically implanted into these distinct microenvironments (Cav-1(+/+) versus Cav-1(-/-) age-matched young female mice). Mammary tumors grown in a Cav-1-deficient tumor microenvironment have an increased stromal content, with vimentin-positive myofibroblasts (a marker associated with oxidative stress) that are also positive for S6-kinase activation (a marker associated with aging). Mammary tumors grown in a Cav-1-deficient tumor microenvironment were more than fivefold larger than tumors grown in a wild-type microenvironment. Thus, a Cav-1-deficient tumor microenvironment provides a fertile soil for breast cancer tumor growth. Interestingly, the mammary tumor-promoting effects of a Cav-1-deficient microenvironment were estrogen and progesterone independent. In this context, chemoprevention was achieved by using the mammalian target of rapamycin (mTOR) inhibitor and anti-aging drug, rapamycin. Systemic rapamycin treatment of mammary tumors grown in a Cav-1-deficient microenvironment significantly inhibited their tumor growth, decreased their stromal content, and reduced the levels of both vimentin and phospho-S6 in Cav-1-deficient cancer-associated fibroblasts. Since stromal loss of Cav-1 is a marker of a lethal tumor microenvironment in breast tumors, these high-risk patients might benefit from treatment with mTOR inhibitors, such as rapamycin or other rapamycin-related compounds (rapalogues).

Objective We studied the expression and function of an mRNA-binding protein, Zinc Finger Protein 36 (ZFP36), in vascular endothelial cells in vivo and in vitro. We tested the hypotheses that ZFP36 regulates inflammation in vascular endothelial cells and that it functions through direct binding to target cytokine mRNAs. We also tested whether ZFP36 inhibits Nuclear Factor-κB (NF-κB) mediated transcriptional responses in vascular endothelial cells. Approach and Results ZFP36 was minimally expressed in healthy aorta but was expressed in endothelial cells overlying atherosclerotic lesions in mice and humans. The protein was also expressed in macrophage foam cells of atherosclerosis. ZFP36 was expressed in human aortic endothelial cells in response to bacterial lipopolysaccharide, glucocorticoid, and forskolin but not oxidized low density lipoproteins or angiotensin II. Functional studies demonstrated that ZFP36 reduces the expression of inflammatory cytokines in target cells by two distinct mechanisms: ZFP36 inhibits NF-κB transcriptional activation and also binds to cytokine mRNAs, leading to reduced transcript stability. Conclusions ZFP36 is expressed in vascular endothelial cells and macrophage foam cells where it inhibits the expression of pro-inflammatory mRNA transcripts. The anti-inflammatory effects of ZFP36 in endothelial cells occur via both transcriptional and post-transcriptional mechanisms. Our data suggest that enhancing vascular ZFP36 expression might reduce vascular inflammation.

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Caveolin (Cav)-1 has been involved in the pathogenesis of ischemic injuries. For instance, modulations of Cav-1 expression have been reported in animal models of myocardial infarction and cerebral ischemia-reperfusion. Furthermore, ablation of the Cav-1 gene in mice has been shown to increase the extent of ischemic injury in models of cerebral and hindlimb ischemia. Cav-1 has also been suggested to play a role in myocardial ischemic preconditioning. However, the role of Cav-1 in myocardial ischemia (MI)-induced cardiac dysfunction still remains to be determined. We determined the outcome of a permanent left anterior descending coronary artery (LAD) ligation in Cav-1 knockout (KO) mice. Wild-type (WT) and Cav-1 KO mice were subjected to permanent LAD ligation for 24 h. The progression of ischemic injury was monitored by echocardiography, hemodynamic measurements, 2,3,5-triphenyltetrazolium chloride staining, β-binding analysis, cAMP level measurements, and Western blot analyses. Cav-1 KO mice subjected to LAD ligation display reduced survival compared with WT mice. Despite similar infarct sizes, Cav-1 KO mice subjected to MI showed reduced left ventricular (LV) ejection fraction and fractional shortening as well as increased LV end-diastolic pressures compared with their WT counterparts. Mechanistically, Cav-1 KO mice subjected to MI exhibit reduced β-adrenergic receptor density at the plasma membrane as well as decreased cAMP levels and PKA phosphorylation. In conclusion, ablation of the Cav-1 gene exacerbates cardiac dysfunction and reduces survival in mice subjected to MI. Mechanistically, Cav-1 KO mice subjected to LAD ligation display abnormalities in β-adrenergic signaling.

Caveolin-1 (Cav-1) is a critical regulator of tumor progression in a variety of cancers where it has been shown to act as either a tumor suppressor or tumor promoter. In glioblastoma multiforme, it has been previously demonstrated to function as a putative tumor suppressor. Our studies here, using the human glioblastoma-derived cell line U-87MG, further support the role of Cav-1 as a negative regulator of tumor growth. Using a lentiviral transduction approach, we were able to stably overexpress Cav-1 in U-87MG cells. Gene expression microarray analyses demonstrated significant enrichment in gene signatures corresponding to downregulation of MAPK, PI3K/AKT and mTOR signaling, as well as activation of apoptotic pathways in Cav-1-overexpressing U-87MG cells. These same gene signatures were later confirmed at the protein level in vitro. To explore the ability of Cav-1 to regulate tumor growth in vivo, we further show that Cav-1-overexpressing U-87MG cells display reduced tumorigenicity in an ectopic xenograft mouse model, with marked hypoactivation of MAPK and PI3K/mTOR pathways. Finally, we demonstrate that Cav-1 overexpression confers sensitivity to the most commonly used chemotherapy for glioblastoma, temozolomide. In conclusion, Cav-1 negatively regulates key cell growth and survival pathways and may be an effective biomarker for predicting response to chemotherapy in glioblastoma.

Obesity is associated with adipose tissue inflammation that contributes to insulin resistance. Zinc finger protein 36 (Zfp36) is an mRNA-binding protein that reduces inflammation by binding to cytokine transcripts and promoting their degradation. We hypothesized that myeloid-specific deficiency of Zfp36 would lead to increased adipose tissue inflammation and reduced insulin sensitivity in diet-induced obese mice. As expected, wild-type (Control) mice became obese and diabetic on a high-fat diet, and obese mice with myeloid-specific loss of Zfp36 [knockout (KO)] demonstrated increased adipose tissue and liver cytokine mRNA expression compared with Control mice. Unexpectedly, in glucose tolerance testing and hyperinsulinemic-euglycemic clamp studies, myeloid Zfp36 KO mice demonstrated improved insulin sensitivity compared with Control mice. Obese KO and Control mice had similar macrophage infiltration of the adipose depots and similar peripheral cytokine levels, but lean and obese KO mice demonstrated increased Kupffer cell (KC; the hepatic macrophage)-expressed Mac2 compared with lean Control mice. Insulin resistance in obese Control mice was associated with enhanced Zfp36 expression in KCs. Compared with Control mice, KO mice demonstrated increased hepatic mRNA expression of a multitude of classical (M1) inflammatory cytokines/chemokines, and this M1-inflammatory hepatic milieu was associated with enhanced nuclear localization of IKKβ and the p65 subunit of NF-κB. Our data confirm the important role of innate immune cells in regulating hepatic insulin sensitivity and lipid metabolism, challenge-prevailing models in which M1 inflammatory responses predict insulin resistance, and indicate that myeloid-expressed Zfp36 modulates the response to insulin in mice.