Ca2+-mediated activation of ERK in hepatocytes by norepinephrine and prostaglandin F2 alpha: role of calmodulin and Src kinases.

BACKGROUND: Previous studies have shown that several agents that stimulate heptahelical G-protein coupled receptors activate the extracellular signal regulated kinases ERK1 (p44mapk) and ERK2 (p42mapk) in hepatocytes. The molecular pathways that convey their signals to ERK1/2 are only partially clarified. In the present study we have explored the role of Ca2+ and Ca2+-dependent steps leading to ERK1/2 activation induced by norepinephrine and prostaglandin (PG)F2alpha. RESULTS: Pretreatment of the cells with the Ca2+ chelators BAPTA-AM or EGTA, as well as the Ca2+ influx inhibitor gadolinium, resulted in a partial decrease of the ERK response. Furthermore, the calmodulin antagonists W-7, trifluoperazine, and J-8 markedly decreased ERK activation. Pretreatment with KN-93, an inhibitor of the multifunctional Ca2+/calmodulin-dependent protein kinase, had no effect on ERK activation. The Src kinase inhibitors PP1 and PP2 partially diminished the ERK responses elicited by both norepinephrine and PGF2alpha. CONCLUSION: The present data indicate that Ca2+ is involved in ERK activation induced by hormones acting on G protein-coupled receptors in hepatocytes, and suggest that calmodulin and Src kinases might play a role in these signaling pathways.

Pyroglutamyl-histidyl-glycine, the endogenous colon mitosis inhibitor, regulates cyclic AMP level in non-tumorigenic colonic epithelial cells.

BACKGROUND: We have proposed that the mitosis inhibiting peptide, pyroGlu-His-Gly (pEHG), a colon-specific negative feedback regulator of cell proliferation, works through a G protein-coupled receptor (GPCR), as do many other pyroglutamyl-peptides. MATERIALS AND METHODS: Non-tumorigenic YAMC (colon mucosa of Immorto mice), IMCE (Immorto-Min mouse hybrid) and human hepatoma (HepG2) cell lines were exposed to pEHG. cAMP concentrations were measured with a protein binding assay, mRNA levels with real-time PCR and Ca2+ concentration with an inverted fluorescence microscope on Fura-2/AM-loaded cells. RESULTS: pEHG (1 nM) increased the intracellular concentration of cAMP after 5-10 min in YAMC cells, but not in HepG2 cells. No effect was seen on cytosolic Ca2+, or in the expression of the proliferation and differentiation regulatory genes c-fos, egr-1 or fosB in YAMC or IMCE cells. CONCLUSION: pEHG stimulates the second messenger cAMP, but has no effect on intracellular Ca2+ or the gene expression of c-fos, egr-1 or fosB.

DHA induces ER stress and growth arrest in human colon cancer cells: associations with cholesterol and calcium homeostasis.

Polyunsaturated fatty acids (PUFAs) are normal constituents of the diet, but have properties different from other fatty acids (e.g., through generation of signaling molecules). N-3 PUFAs reduce cancer cell growth, but no unified mechanism has been identified. We show that docosahexaenoic acid (DHA; 22:6 n-3) causes extensive changes in gene expression patterns at mRNA level in the colon cancer cell line SW620. Early changes include unfolded protein response (UPR) and increased levels of phosphorylated eIF2alpha as verified at protein level. The latter is considered a hallmark of endoplasmic reticulum (ER) stress and is abundantly present already after 3 h. It may coordinate many of the downstream changes observed, including signaling pathways for cell cycle arrest/apoptosis, calcium homeostasis, cholesterol metabolism, ubiquitination, and proteasomal degradation. Also, eicosapentaenoic acid (EPA), but not oleic acid (OA), induced key mediators of ER stress and UPR at protein level. Accumulation of esterified cholesterol was not compensated for by increased total levels of cholesterol, and mRNAs for cholesterol biosynthesis as well as de novo synthesis of cholesterol were reduced. These results suggest that cytotoxic effects of DHA are associated with signaling pathways involving lipid metabolism and ER stress.

Intercellular calcium signalling in cultured renal epithelia: a theoretical study of synchronization mode and pacemaker activity.

We investigate a two-dimensional lattice model representation of intercellular Ca2+ signalling in a population of epithelial cells coupled by gap junctions. The model is based on and compared with Ca2+ imaging data from globally bradykinin-stimulated MDCK-I (Madin-Darby canine kidney)-I cell layers. We study large-scale synchronization of relevance to our laboratory experiments. The system is found to express a wealth of dynamics, including quasiperiodic, chaotic and multiply-periodic behaviour for intermediate couplings. We take a particular interest in understanding the role of "pacemaker cells" in the synchronization process. It has been hypothesized that a few highly hormone-sensitive cells control the collective frequency of oscillation, which is close to the natural frequencies (without coupling) of these cells. The model behaviour is consistent with the conjectures of the pacemaker cell hypothesis near the critical coupling where the cells lock onto a single frequency. However, the simulations predict that the frequency in globally connected systems decreases with increasing coupling. It is found that a pacemaker is not defined by its natural frequency alone, but that other intrinsic or local factors must be considered. Inclusion of partly sensitized cells that do not oscillate autonomously in the cell layer increases the coupling necessary for global synchronization. For not excessively high coupling, these cells oscillate irregularly and with distinctive lower frequencies. In summary, the present study shows that the frequency of synchronized oscillations is not dictated by one or few fast-responding cells. The collective frequency is the result of a two-way communication between the phase-advanced pacemaker and its environment.

Antibody-mediated delivery of antigen to chemokine receptors on antigen-presenting cells results in enhanced CD4+ T cell responses.

Here, we have investigated if targeting of T cell epitopes to chemokine receptors results in improved CD4+ T cell responses. Mouse monoclonal antibodies (mAb) with kappaL chains were targeted to various chemokine receptors expressed on human monocytes or immature dendritic cells (DC), and proliferation of cloned human, DR4-restricted CD4+ T cells specific for mouse Ckappa(40-48) was measured. When using monocytes as antigen-presenting cells, mAb specific for CCR1, CCR2, CCR5, and CXCR4 were 100-10,000-fold more efficient at inducing T cell proliferation when compared to isotype-matched control mAb on a per molecule basis. Targeting of immature DC was less effective and was only seen with anti-CCR1 and anti-CXCR4 mAb. Anti-chemokine receptors mAb required to be processed by the conventional endosomal MHC class II presentation pathway. The mAb did not induce signaling through the chemokine receptors as they failed to induce mobilization of cytosolic Ca2+ and actin polymerization. They also failed to induce APC maturation. The results strongly suggest that chemokine receptors channel antigen into the endocytic pathway for presentation on MHC class II molecules. Targeting T cell epitopes to chemokine receptors by recombinant antibody should be a useful vaccine strategy for the induction of strong CD4+ T cell responses.

Cutting edge: link between innate and adaptive immunity: Toll-like receptor 2 internalizes antigen for presentation to CD4+ T cells and could be an efficient vaccine target.

An ideal vaccine for induction of CD4(+) T cell responses should induce local inflammation, maturation of APC, and peptide loading of MHC class II molecules. Ligation of Toll-like receptor (TLR) 2 provides the first two of these three criteria. We have studied whether targeting of TLR2 results in loading of MHC class II molecules and enhancement of CD4(+) T cell responses. To dissociate MHC class II presentation from APC maturation, we have used an antagonistic, mouse anti-human TLR2 mAb (TL2.1) as ligand and measured proliferation of a mouse Ckappa-specific human CD4(+) T cell clone. TL2.1 mAb was 100-1000 times more efficiently presented by APC compared with isotype-matched control mAb. Moreover, TL2.1 mAb was internalized into endosomes and processed by the conventional MHC class II pathway. This novel function of TLR2 represents a link between innate and adaptive immunity and indicates that TLR2 could be a promising target for vaccines.