Cholesterol-load evokes robust calcium response in macrophages: An early event toward cholesterol-induced macrophage death.

Macrophages in atherosclerotic lesions accumulate large amounts of unesterified cholesterol. Excess cholesterol load leads to cell death of macrophages, which is associated with the progression of atherosclerotic lesions. Calcium depletion in the endoplasmic reticulum (ER) and subsequent pro-apoptotic aberrant calcium signaling are key events in cholesterol-induced macrophage death. Although these concepts imply cytoplasmic calcium events in cholesterol-loaded macrophages, the mechanisms linking cholesterol accumulation to cytoplasmic calcium response have been poorly investigated. Based on our previous finding that extracellularly applied cholesterol evoked robust calcium oscillations in astrocytes, a type of glial cells in the brain, we hypothesized that cholesterol accumulation in macrophages triggers cytoplasmic calcium elevation. Here, we showed that cholesterol application induces calcium transients in THP-1-derived and peritoneal macrophages. Inhibition of inositol 1,4,5-trisphosphate receptors (IP3Rs) and l-type calcium channels (LTCCs) prevented cholesterol-induced calcium transients and ameliorated cholesterol-induced macrophage death. These results suggest that cholesterol-induced calcium transients through IP3Rs and LTCCs are crucial mechanisms underlying cholesterol-induced cell death of macrophages.

Cholesterol-induced robust Ca oscillation in astrocytes required for survival and lipid droplet formation in high-cholesterol condition.

Cholesterol, one of the major cell membrane components, stabilizes membrane fluidity and regulates signal transduction. Beside its canonical roles, cholesterol has been reported to directly activate signaling pathways such as hedgehog (Hh). We recently found that astrocytes, one of the glial cells, respond to Hh pathway stimulation by Ca signaling. These notions led us to test if extracellularly applied cholesterol triggers Ca signaling in astrocytes. Here, we found that cholesterol application induces robust Ca oscillation only in astrocytes with different properties from the Hh-induced Ca response. The Ca oscillation has a long delay which corresponds to the onset of cholesterol accumulation in the plasma membrane. Blockade of the Ca oscillation resulted in enhancement of astrocytic cell death and disturbance of lipid droplet formation, implying a possibility that the cholesterol-induced Ca oscillation plays important roles in astrocytic survival and cholesterol handling under pathological conditions of cholesterol load such as demyelination.

Proliferative Classification of Intracranially Injected HER2-positive Breast Cancer Cell Lines.

HER2 is overexpressed in 25-30% of breast cancers, and approximately 30% of HER2-positive breast cancers metastasize to the brain. Although the incidence of brain metastasis in HER2-positive breast cancer is high, previous studies have been mainly based on cell lines of the triple-negative subtype, and the molecular mechanisms of brain metastasis in HER2-positive breast cancer are unclear. In the present study, we performed intracranial injection using nine HER2-positive breast cancer cell lines to evaluate their proliferative activity in brain tissue. Our results show that UACC-893 and MDA-MB-453 cells rapidly proliferated in the brain parenchyma, while the other seven cell lines moderately or slowly proliferated. Among these nine cell lines, the proliferative activity in brain tissue was not correlated with either the HER2 level or the HER2 phosphorylation status. To extract signature genes associated with brain colonization, we conducted microarray analysis and found that these two cell lines shared 138 gene expression patterns. Moreover, some of these genes were correlated with poor prognosis in HER2-positive breast cancer patients. Our findings might be helpful for further studying brain metastasis in HER2-positive breast cancer.

Sonic hedgehog enhances calcium oscillations in hippocampal astrocytes.

Sonic hedgehog (SHH) is important for organogenesis during development. Recent studies have indicated that SHH is also involved in the proliferation and transformation of astrocytes to the reactive phenotype. However, the mechanisms underlying these are unknown. Involvement of SHH signaling in calcium (Ca) signaling has not been extensively studied. Here, we report that SHH and Smoothened agonist (SAG), an activator of the signaling receptor Smoothened (SMO) in the SHH pathway, activate Ca oscillations in cultured murine hippocampal astrocytes. The response was rapid, on a minute time scale, indicating a noncanonical pathway activity. Pertussis toxin blocked the SAG effect, indicating an involvement of a Gi coupled to SMO. Depletion of extracellular ATP by apyrase, an ATP-degrading enzyme, inhibited the SAG-mediated activation of Ca oscillations. These results indicate that SAG increases extracellular ATP levels by activating ATP release from astrocytes, resulting in Ca oscillation activation. We hypothesize that SHH activates SMO-coupled Gi in astrocytes, causing ATP release and activation of Gq/11-coupled P2 receptors on the same cell or surrounding astrocytes. Transcription factor activities are often modulated by Ca patterns; therefore, SHH signaling may trigger changes in astrocytes by activating Ca oscillations. This enhancement of Ca oscillations by SHH signaling may occur in astrocytes in the brain in vivo because we also observed it in hippocampal brain slices. In summary, SHH and SAG enhance Ca oscillations in hippocampal astrocytes, Gi mediates SAG-induced Ca oscillations downstream of SMO, and ATP-permeable channels may promote the ATP release that activates Ca oscillations in astrocytes.