Novel anti-inflammatory mechanisms of N-Acetyl-Ser-Asp-Lys-Pro in hypertension-induced target organ damage

High blood pressure (HBP) is an important risk factor for cardiac, renal, and vascular dysfunction. Excess inflammation is the major pathogenic mechanism for HBP-induced target organ damage (TOD). N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP), a tetrapeptide specifically degraded by angiotensin converting enzyme (ACE), reduces inflammation, fibrosis, and TOD induced by HBP. Our hypothesis is that Ac-SDKP exerts its anti-inflammatory effects by inhibiting: 1) differentiation of bone marrow stem cells (BMSC) to macrophages, 2) activation and migration of macrophages, and 3) release of the proinflammatory cytokine TNF-alpha by activated macrophages. BMSC were freshly isolated and cultured in macrophage growth medium. Differentiation of murine BMSC to macrophages was analyzed by flow cytometry using F4/80 as a marker of macrophage maturation. Macrophage migration was measured in a modified Boyden chamber. TNF-alpha release by activated macrophages in culture was measured by ELISA. Myocardial macrophage activation in mice with ANG II-induced hypertension was studied by Western blotting of Mac-2 (galectin-3) protein. Interstitial collagen deposition was measured by picrosirius red staining. We found that Ac-SDKP (10 nM) reduced differentiation of cultured BMSC to mature macrophages by 24.5% [F4/80 positivity: 14.09 +/- 1.06 mean fluorescent intensity for vehicle and 10.63 +/- 0.35 for Ac-SDKP; P < 0.05]. Ac-SDKP also decreased galectin-3 and macrophage colony-stimulating factor-dependent macrophage migration. In addition, Ac-SDKP decreased secretion of TNF-alpha by macrophages stimulated with bacterial LPS. In mice with ANG II-induced hypertension, Ac-SDKP reduced expression of galectin-3, a protein produced by infiltrating macrophages in the myocardium, and interstitial collagen deposition. In conclusion, this study demonstrates that part of the anti-inflammatory effect of Ac-SDKP is due to its direct effect on BMSC and macrophage, inhibiting their differentiation, activation, and cytokine release. These effects explain some of the anti-inflammatory and antifibrotic properties of Ac-SDKP in hypertension.     

Lactobacillus gasseri in the Upper Small Intestine Impacts an ACSL3-Dependent Fatty Acid-Sensing Pathway Regulating Whole-Body Glucose Homeostasis

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Quantitative phosphoproteomic analysis reveals reciprocal activation of receptor tyrosine kinases between cancer epithelial cells and stromal fibroblasts

Background

Cancer-associated fibroblasts (CAFs) are one of the most important components of tumor stroma and play a key role in modulating tumor growth. However, a mechanistic understanding of how CAFs communicate with tumor cells to promote their proliferation and invasion is far from complete. A major reason for this is that most current techniques and model systems do not capture the complexity of signal transduction that occurs between CAFs and tumor cells.

Methods

In this study, we employed a stable isotope labeling with amino acids in cell culture (SILAC) strategy to label invasive breast cancer cells, MDA-MB-231, and breast cancer patient-derived CAF cells. We used an antibody-based phosphotyrosine peptide enrichment method coupled to LC–MS/MS to catalog and quantify tyrosine phosphorylation-mediated signal transduction events induced by the bidirectional communication between patient-derived CAFs and tumor cells.

Results

We discovered that distinct signaling events were activated in CAFs and in tumor epithelial cells during the crosstalk between these two cell types. We identified reciprocal activation of a number of receptor tyrosine kinases including EGFR, FGFR1 and EPHA2 induced by this bidirectional communication.

Conclusions

Our study not only provides insights into the mechanisms of the interaction between CAFs and tumor cells, but the model system described here could be used as a prototype for analysis of intercellular communication in many different tumor microenvironments.

     

PIK3CA somatic mutations in breast cancer: Mechanistic insights from Langevin dynamics simulations

Somatic mutations in PIK3CA (phosphatidylinositol-3 kinase, catalytic subunit, alpha isoform) are reported in breast and other human cancers to concentrate at hotspots within its kinase and helical domains. Most of these mutations cause kinase gain of function in vitro and are associated with oncogenicity in vivo. However, little is known about the mechanisms driving tumor development. We have performed computational structural studies on a homology model of wildtype PIK3CA plus recurrent H1047R, H1047L, and P539R mutations, located in the kinase and helical domains, respectively. The time evolution of the structures show that H1047R/L mutants exhibit a larger area of the catalytic cleft between the kinase N- and C-lobes compared with the wildtype that could facilitate the entrance of substrates. This larger area might yield enhanced substrate-to-product turnover associated with oncogenicity. In addition, the H1047R/L mutants display increased kinase activation loop mobility, compared with the wildtype. The P539R mutant forms more hydrogen bonds and salt-bridge interactions than the wildtype, properties that are associated with enhanced thermostability. Mutant-specific differences in the catalytic cleft and activation loop behavior suggest that structure-based mutant-specific inhibitors can be designed for PIK3CA-positive breast cancers.     

Lecanicillium and Verticillium species from Indonesia and Japan including three new species

Forty-six Lecanicillium strains and one Verticillium strain were isolated from subterranean and epiphytic arthropods, soil, and other sources collected in Indonesia and Japan. These strains were identified as nine Lecanicillium and one Verticillium species including six undescribed species based on light microscopy and the sequences of the ITS-1 and ITS-2 regions including 5.8S ribosomal DNA. Four of the ten species (L. araneicola, L. kalimantanense, Lecanicillium sp. 4, and V. indonesiacum) were recovered from Indonesia, five of the ten (L. attenuatum, L. fusisporum, L. psalliotae, Lecanicillium sp. 1, and Lecanicillium sp. 3) were from Japan, and L. saksenae was from both countries. In this article, new species (L. araneicola, L. kalimantanense, and V. indonesiacum) and a new combination (L. saksenae) are proposed from the fungi isolated from epiphytic and subterranean arthropods collected in East Kalimantan.     

Store-independent activation of Orai1 by SPCA2 in mammary tumors

Ca(2+) is an essential and ubiquitous second messenger. Changes in cytosolic Ca(2+) trigger events critical for tumorigenesis, such as cellular motility, proliferation, and apoptosis. We show that an isoform of Secretory Pathway Ca(2+)-ATPase, SPCA2, is upregulated in breast cancer-derived cells and human breast tumors, and suppression of SPCA2 attenuates basal Ca(2+) levels and tumorigenicity. Contrary to its conventional role in Golgi Ca(2+) sequestration, expression of SPCA2 increased Ca(2+) influx by a mechanism dependent on the store-operated Ca(2+) channel Orai1. Unexpectedly, SPCA2-Orai1 signaling was independent of ER Ca(2+) stores or STIM1 and STIM2 sensors and uncoupled from Ca(2+)-ATPase activity of SPCA2. Binding of the SPCA2 amino terminus to Orai1 enabled access of its carboxyl terminus to Orai1 and activation of Ca(2+) influx. Our findings reveal a signaling pathway in which the Orai1-SPCA2 complex elicits constitutive store-independent Ca(2+) signaling that promotes tumorigenesis.