Estrogen Regulation of mTOR Signaling and Mitochondrial Function in Invasive Lobular Carcinoma Cell Lines Requires WNT4.

Invasive lobular carcinoma of the breast (ILC) is strongly estrogen-driven and represents a unique context for estrogen receptor (ER) signaling. In ILC, ER controls the expression of the Wnt ligand WNT4, which is critical for endocrine response and anti-estrogen resistance. However, signaling mediated by WNT4 is cell type- and tissue-specific, and has not been explored in ILC. We utilized reverse phase protein array (RPPA) to characterize ER and WNT4-driven signaling in ILC cells and identified that WNT4 mediates downstream mTOR signaling via phosphorylation of S6 Kinase. Additionally, ER and WNT4 control levels of MCL-1, which is associated with regulation of mitochondrial function. In this context, WNT4 knockdown led to decreased ATP production and increased mitochondrial fragmentation. WNT4 regulation of both mTOR signaling and MCL-1 were also observed in anti-estrogen resistant models of ILC. We identified that high WNT4 expression is associated with similar mTOR pathway activation in ILC and serous ovarian cancer tumors, suggesting that WNT4 signaling is active in multiple tumor types. The identified downstream pathways offer insight into WNT4 signaling and represent potential targets to overcome anti-estrogen resistance for patients with ILC.

Wnt family member 4 (WNT4) and WNT3A activate cell-autonomous Wnt signaling independent of porcupine O-acyltransferase or Wnt secretion.

Porcupine O-acyltransferase (PORCN) is considered essential for Wnt secretion and signaling. However, we observed that PORCN inhibition does not phenocopy the effects of WNT4 knockdown in WNT4-dependent breast cancer cells. This suggests a unique relationship between PORCN and WNT4 signaling. To examine the role of PORCN in WNT4 signaling, here we overexpressed WNT4 or WNT3A in breast cancer, ovarian cancer, and fibrosarcoma cell lines. Conditioned media from these lines and co-culture systems were used to assess the dependence of Wnt secretion and activity on the critical Wnt secretion proteins PORCN and Wnt ligand secretion (WLS) mediator. We observed that WLS is universally required for Wnt secretion and paracrine signaling. In contrast, the dependence of WNT3A secretion and activity on PORCN varied across the cell lines, and WNT4 secretion was PORCN-independent in all models. Surprisingly, WNT4 did not exhibit paracrine activity in any tested context. Absent the expected paracrine activity of secreted WNT4, we identified cell-autonomous Wnt signaling activation by WNT4 and WNT3A, independent of PORCN or Wnt secretion. The PORCN-independent, cell-autonomous Wnt signaling demonstrated here may be critical in WNT4-driven cellular contexts or in those that are considered to have dysfunctional Wnt signaling.

Mice Lacking Fatty Acid-Binding Protein 5 Are Resistant to Listeria monocytogenes.

To investigate the role of fatty acid-binding protein 5 (FABP5) in infectious diseases, FABP5-deficient mice were challenged with Listeria monocytogenes, a facultative intracellular bacterial pathogen. Interestingly, FABP5-deficient animals were able to clear the infection within 3 days whereas control wild-type (WT) animals showed comparatively higher bacterial burdens in the liver and spleen. Sections of infected tissues showed an increase in inflammatory foci in WT mice compared to FABP5-deficient mice. FABP5-deficient mice had lower circulating inflammatory cytokines and increased inducible nitric oxide synthase production. FABP5-deficient mouse bone marrow-derived macrophages produced higher levels of nitrite anion than their WT counterparts in response to various stimuli. Additionally, in contrast to FABP5-/- mice, transgenic mice overexpressing FABP5 in myeloid cells (LysM-Cre driven) showed decreased survival rates and increased bacterial burden and inflammatory cytokines. Overall, these findings suggest that increased FABP5 levels correlate with a higher L. monocytogenes bacterial burden and elevated subsequent inflammation.

Impact of fatty acid binding protein 5-deficiency on COPD exacerbations and cigarette smoke-induced inflammatory response to bacterial infection.

BACKGROUND: Although cigarette smoking (CS) is by far the most important risk factor of chronic obstructive pulmonary disease (COPD), repeated and sustained infections are clearly linked to disease pathogenesis and are responsible for acute inflammatory flares (i.e. COPD exacerbations). We have previously identified Fatty Acid Binding Protein 5 (FABP5) as an important anti-inflammatory protein in primary airway epithelial cells. RESULTS: In this study we found decreased FABP5 mRNA and protein levels in peripheral blood mononuclear cells (PBMCs) of COPD patients, especially among those who reported episodes of COPD exacerbations. Using wildtype (WT) and FABP5-/- mice, we examined the effects of FABP5 on CS and infection-induced inflammatory responses. Similarly to what we saw in airway epithelial cells, infection increased FABP5 expression while CS decreased FABP5 expression in mouse lung tissues. CS-exposed and P. aeruginosa-infected FABP5-/- mice had significantly increased inflammation as shown by increased lung histopathological score, cell infiltration and inflammatory cytokine levels. Restoration of FABP5 in alveolar macrophages using a lentiviral approach attenuated the CS- and bacteria-induced pulmonary inflammation. And finally, while P. aeruginosa infection increased PPARγ activity, CS or FABP5 knockdown greatly reduced PPARγ activity. CONCLUSIONS: These findings support a model in which CS-induced FABP5 inhibition contributes to increased inflammation in COPD exacerbations. It is interesting to speculate that the increased inflammation is a result of decreased PPARγ activity.

The ion channel, TRPM2, contributes to the pathogenesis of radiodermatitis.

Radiodermatitis is a painful side effect for cancer patients undergoing radiotherapy. Irradiation of the skin causes inflammation and breakdown of the epidermis and can lead to significant morbidity and mortality in severe cases, as seen in exposure from accidents or weapons such as "dirty bombs" and ultimately leads to tissue fibrosis. However, the pathogenesis of radiodermatitis is not fully understood. Using a mouse model of radiodermatitis, we showed that the Transient Receptor Potential Melastatin 2 (TRPM2) ion channel plays a significant role in the development of dermatitis following exposure to ionizing radiation. Irradiated TRPM2-deficient mice developed less inflammation, fewer severe skin lesions and decreased fibrosis when compared to wild type mice. The TRPM2-deficient mice also showed a faster recovery period as seen by their increased weight gain post irradiation. Finally, TRPM2-deficient mice exhibited lower systemic inflammation with a reduction in inflammatory cytokines present in the serum. These findings suggest that TRPM2 may be a potential therapeutic target for reducing the severity of radiodermatitis.

The TRPM2 ion channel contributes to cytokine hyperproduction in a mouse model of Down Syndrome.

Trisomy 21 (Down Syndrome, DS) is the most common chromosomal anomaly. Although DS is mostly perceived as affecting cognitive abilities and cardiac health, individuals with DS also exhibit dysregulated immune functions. Levels of pro-inflammatory cytokines are increased, but intrinsic alterations of innate immunity are understudied in DS. Furthermore, elevated Reactive Oxygen Species (ROS) are well documented in individuals with DS, further exacerbating inflammatory processes. Chronic inflammation and oxidative stress are often precursors of subsequent tissue destruction and pathologies, which affect a majority of persons with DS. Together with ROS, the second messenger ion Ca2+ plays a central role in immune regulation. TRPM2 (Transient Receptor Potential Melastatin 2) is a Ca2+-permeable ion channel that is activated under conditions of oxidative stress. The Trpm2 gene is located on human Chromosome 21 (Hsa21). TRPM2 is strongly represented in innate immune cells, and numerous studies have documented its role in modulating inflammation. We have previously found that as a result of suboptimal cytokine production, TRPM2-/- mice are highly susceptible to the bacterial pathogen Listeria monocytogenes (Lm). We therefore used Lm infection to trigger and characterize immune responsiveness in the DS mouse model Dp10(yey), and to investigate the potential contribution of TRPM2. In comparison to wildtype (WT), Dp10(yey) mice show an increased resistance against Lm infection and higher IFNγ serum concentrations. Using a gene elimination approach, we show that these effects correlate with Trpm2 gene copy number, supporting the notion that Trpm2 might promote hyperinflammation in DS.

TRPM7 Kinase Controls Calcium Responses in Arterial Thrombosis and Stroke in Mice.

OBJECTIVE: TRPM7 (transient receptor potential cation channel, subfamily M, member 7) is a ubiquitously expressed bifunctional protein comprising a transient receptor potential channel segment linked to a cytosolic α-type serine/threonine protein kinase domain. TRPM7 forms a constitutively active Mg2+ and Ca2+ permeable channel, which regulates diverse cellular processes in both healthy and diseased conditions, but the physiological role of TRPM7 kinase remains largely unknown. APPROACH AND RESULTS: Here we show that point mutation in TRPM7 kinase domain deleting the kinase activity in mice (Trpm7R/R ) causes a marked signaling defect in platelets. Trpm7R/R platelets showed an impaired PIP2 (phosphatidylinositol-4,5-bisphosphate) metabolism and consequently reduced Ca2+ mobilization in response to stimulation of the major platelet receptors GPVI (glycoprotein VI), CLEC-2 (C-type lectin-like receptor), and PAR (protease-activated receptor). Altered phosphorylation of Syk (spleen tyrosine kinase) and phospholipase C γ2 and ß3 accounted for these global platelet activation defects. In addition, direct activation of STIM1 (stromal interaction molecule 1) with thapsigargin revealed a defective store-operated Ca2+ entry mechanism in the mutant platelets. These defects translated into an impaired platelet aggregate formation under flow and protection of the mice from arterial thrombosis and ischemic stroke in vivo. CONCLUSIONS: Our results identify TRPM7 kinase as a key modulator of phospholipase C signaling and store-operated Ca2+ entry in platelets. The protection of Trpm7R/R mice from acute ischemic disease without developing intracranial hemorrhage indicates that TRPM7 kinase might be a promising antithrombotic target.

Cigarette smoke inhibits LPS-induced FABP5 expression by preventing c-Jun binding to the FABP5 promoter.

Cigarette smoking is the primary cause of chronic obstructive pulmonary disease (COPD) with repeated and sustained infections linked to disease pathogenesis and exacerbations. The airway epithelium constitutes the first line of host defense against infection and is known to be impaired in COPD. We have previously identified Fatty Acid Binding Protein 5 (FABP5) as an important anti-inflammatory player during respiratory infections and showed that overexpression of FABP5 in primary airway epithelial cells protects against bacterial infection and inflammation. While cigarette smoke down regulates FABP5 expression, its mechanism remains unknown. In this report, we have identified three putative c-Jun binding sites on the FABP5 promoter and show that cigarette smoke inhibits the binding of c-Jun to its consensus sequence and prevents LPS-induced FABP5 expression. Using chromatin immunoprecipitation, we have determined that c-Jun binds the FABP5 promoter when stimulated with LPS but the presence of cigarette smoke greatly reduces this binding. Furthermore, cigarette smoke or a mutation in the c-Jun binding site inhibits LPS-induced FABP5 promoter activity. These data demonstrate that cigarette smoke interferes with FABP5 expression in response to bacterial infection. Thus, functional activation of FABP5 may be a new therapeutic strategy when treating COPD patients suffering from exacerbations.