Dysregulation of intracellular pH is a cause of impaired capacitation in Slc22a14-deficient mice

Solute carrier 22a member 14 (SLC22A14) plays a critical role in male infertility in mice. We previously revealed that one of the causes of infertility is impaired capacitation. However, the molecular mechanism remained unclear. Here, we show that the influx of HCO3–, a trigger of capacitation, is impaired and intracellular pH is decreased in the sperm of Slc22a14 knockout (KO) mice. While intracellular cAMP concentration did not increase during capacitation in Slc22a14 KO spermatozoa, HCO3–-dependent soluble adenylate cyclase activity was normal, and the addition of 8-bromo cAMP rescued the decreased protein tyrosine phosphorylation. In addition, the intracellular pH of Slc22a14 KO sperm was lower than that of wild-type sperm and did not increase after the addition of HCO3–. Although its relationship to the regulation of intracellular pH is unknown, TMEM225, a possible protein phosphatase inhibitor, was found to be decreased in Slc22a14 KO sperm. The decreased in vitro fertilization rate of Slc22a14 KO sperm was partially rescued by an increase in the intracellular pH (pHi) and the addition of 8-bromo cAMP. These results suggest that SLC22A14 is involved in capacitation through the regulation of HCO3– transport and pHi.

Enhancement of DPP-IV Inhibitory Activity and GLP-1 Release Through RADA16-assisted Molecular Designed Rapeseed Peptide Nanogels

Objectives To produce and evaluate the microstructure and rheological behavior of a nanogel of pentapeptide IPQVS (RAP1) and octopeptide ELHQEEPL (RAP2), derived from rapeseed napin, through RADA16-assisted molecular design. In addition, to determine in vitro the dipeptidyl-peptidase IV (DPP-IV) inhibitory properties, glucagon-like peptide-1 (GLP-1) secretion through activation of calcium-sensing receptor (CaSR) and the intracellular calcium ion mobilization and receptor protein cAMP concentration. Methods An innovative nanogel, which loaded the RAP1 and RAP2 into RADA16 scaffold, respectively, was synthesized. The linker of double Gly was used in the connection of RADA16 and the functional oligopeptide region. DPP-IV inhibitory activity was evaluated in the Caco-2 cell monolayer. The microstructure and rheological behavior of RADA16-RAP1 and RADA16-RAP2 were characterized. The GLP-1 secretion through activation of CaSR receptor and the intracellular calcium ion mobilization and cAMP concentration were determined in STC-1 cells. Results DPP-IV inhibitory activity was reduced (more potent) by over 65% in the presence of 250 μM of the two formed nanogels RADA16–GG-IPQVS and RADA16- GG-ELHQEEPL (p < 0.05). The two nanogel peptides had high stability at low temperature or body temperature and high dispersibility in water. Stable β-sheet structures increased by 5.6-fold and 5.2-fold, respectively, than the original oligopeptides, with a self-assembled fibrous morphology. The nanogels RADA16-RAP1 and RADA16-RAP2 might exhibit good rheological properties for potential injectable applications; storage modulus (G’) was 10 times higher than low modulus (G’’). Furthermore, the RAP2 and its RADA16-assisted nanogel peptide at the concentration of 250 μM significantly (p < 0.05) increased the release of GLP-1 by 35.46% through calcium-sensing receptor (CaSR) pathway in the enteroendocrine STC-1 cells. Conclusions The innovated nanogel with the sequence of RADA16-GG-Xn has the possibility of oral and injection to management type 2 diabetes. DPP-IV inhibitory peptides IPQVS and ELHQEEPL can form a nanogel with high water content through the self-assembly gelation of RADA16. They may attach to the brush border of human intestinal epithelial cells to inhibit DPP-IV in the intestinal lumen. Funding Sources USDA-HATCH [grant number 1017440].

Inhibition of cAMP/PKA/CREB Signaling Axis Improves Fibroblast Plasticity for Direct Cardiac Reprogramming

Direct cardiac reprogramming of fibroblasts into induced cardiomyocytes (iCMs) is a promising approach but remains a challenging technology of regenerative medicine for damaged myocardium. Efforts have been focused on improving the efficiency by understanding fundamental mechanisms. One of the major challenges is that the plasticity of cultured fibroblast varies batch to batch with unknown mechanisms. Here, we noticed that a portion of in vitro cultured fibroblasts have been activated to differentiate into myofibroblasts, marked by the expression of αSMA, even in the primary cell culture of tissues. Both forskolin, which activates adenylyl cyclase and increases cAMP concentration, and TGFbeta inhibitor SB431542 can efficiently suppress myofibroblast differentiation of cultured fibroblasts. However, SB431542 improved but forskolin blocked iCM reprogramming of fibroblasts that were infected with retroviruses of Gata4, Mef2c and Tbx5 (GMT). Moreover, inhibitors of cAMP downstream signaling pathways, PKA or CREB-CBP, significantly improved the efficiency of iCM reprogramming. Consistently, inhibition of p38/MAPK, another upstream regulator of CREB-CBP, also improved reprogramming efficiency. We then investigated if inhibition of these signaling pathways in primary cultured fibroblast could improve their plasticity for reprogramming, and found that preconditioning of cultured fibroblasts with CREB-CBP inhibitor significantly improved the cellular plasticity of fibroblasts to be reprogrammed, yielding ~2-fold amount of reprogrammed iCMs compared to that of untreated control cells. In conclusion, suppression of cAMP/PKA/CREB signaling axis improves fibroblast plasticity for direct cardiac reprogramming.

Dual Inhibition of Ornithine Decarboxylase and A1 Adenosine Receptor Efficiently Suppresses Breast Tumor Cells

Personized treatment of breast cancer is still a challenge, and more treatment options for breast cancer are warranted. Combination therapies have been a highly appreciated strategy for breast cancer treatment in recent years, and the development of new combination therapies could improve patient outcomes. Adenosine and polyamines are both endogenous metabolites with indispensable biological functions. Adenosine binds with the A1 adenosine receptor (A1AR) to downregulate cAMP concentration, and both low cAMP content and high polyamine levels stimulate the growth and proliferation of cancer cells. In this work, we initially used a polyamine synthesis inhibitor, DFMO (α-difluoromethylornithine), and an A1AR inhibitor, DPCPX (8-cyclopentyl-1,3-dipropylxanthine) to investigate if simultaneously inhibiting A1AR and polyamine synthesis has synergistical antitumor effects. Next, we investigated a dual inhibitor (ODC-MPI-2) of A1AR and ODC (ornithine decarboxylase 1), the rate-limiting enzyme in polyamine biosynthesis. We investigated if ODC-MPI-2 could inhibit the proliferation and growth of breast cancer cells. Our data showed that DFMO and DPCPX synergistically inhibit the growth and proliferation of MCF-7 cells. We also demonstrated that ODC-MPI-2 reduces cellular polyamine levels and elevates cAMP concentration. We further showed that ODC-MPI-2 inhibits the growth, proliferation, and migration/invasion of MCF-7 cells. Finally, ODC-MPI-2 showed a preference for inhibiting triple-negative breast cancer cells. The dual inhibition of ODC and A1AR is a new combination therapy strategy for treating breast cancer, and dual inhibitors of ODC and A1AR may be effective future drugs for treating breast cancer.

Glycine Cleavage System and cAMP Receptor Protein Co-Regulate CRISPR/cas3 Expression to Resist Bacteriophage

The CRISPR/Cas system protects bacteria against bacteriophage and plasmids through a sophisticated mechanism where cas operon plays a crucial role consisting of cse1 and cas3. However, comprehensive studies on the regulation of cas3 operon of the Type I-E CRISPR/Cas system are scarce. Herein, we investigated the regulation of cas3 in Escherichia coli. The mutation in gcvP or crp reduced the CRISPR/Cas system interference ability and increased bacterial susceptibility to phage, when the casA operon of the CRISPR/Cas system was activated. The silence of the glycine cleavage system (GCS) encoded by gcvTHP operon reduced cas3 expression. Adding N5, N10-methylene tetrahydrofolate (N5, N10-mTHF), which is the product of GCS-catalyzed glycine, was able to activate cas3 expression. In addition, a cAMP receptor protein (CRP) encoded by crp activated cas3 expression via binding to the cas3 promoter in response to cAMP concentration. Since N5, N10-mTHF provides one-carbon unit for purine, we assumed GCS regulates cas3 through associating with CRP. It was evident that the mutation of gcvP failed to further reduce the cas3 expression with the crp deletion. These results illustrated a novel regulatory pathway which GCS and CRP co-regulate cas3 of the CRISPR/Cas system and contribute to the defence against invasive genetic elements, where CRP is indispensable for GCS regulation of cas3 expression.

Biphasic Response of Protein Kinase A to Cyclic Adenosine Monophosphate Triggers Distinct Epithelial Phenotypes

Despite the large diversity of the proteins involved in cellular signaling, many intracellular signaling pathways converge onto one of only dozens of small molecule second messengers. Cyclic adenosine monophosphate (cAMP), one of these second messengers, is known to regulate activity of both Protein Kinase A (PKA) and the Extracellular Regulated Kinase (ERK), among other signaling pathways. In its role as an important cellular signaling hub, intracellular cAMP concentration has long been assumed to monotonically regulate its known effectors.Using an optogenetictool that can introduce precise amounts of cAMP in MDCKI cells, we identify genes whose expression changes biphasically with monotonically increasing cAMP levels. By examining the behavior of PKA and ERK1/2 in the same dose regime, we find that these kinases also respond biphasically to increasing cAMP levels, with opposite phases. We reveal that this behavior results from an elaborate integration by PKA of many cellular signals triggered by cAMP. In addition to the direct activation of PKA, cAMP also modulates the activity of p38 and ERK, which then converge to inhibit PKA. These interactions and their ensuing biphasic PKA profile have important physiological repercussions, influencing the ability of MDCKI cells to proliferate and form acini. Our data, supported by computational modeling, synthesize a set of network interconnections involving PKA and other important signaling pathways into a model that demonstrates how cells can capitalize on signal integration to create a diverse set of responses to cAMP concentration and produce complex input-output relationships.

Elevated intracellular cAMP concentration mediates growth suppression in glioma cells

SUMMARYSupressed levels of intracellular cAMP have been associated with malignancy. Thus, elevating cAMP through activation of adenylyl cyclase (AC) or by inhibition of phosphodiesterase (PDE) may be therapeutically beneficial. Here, we demonstrate that elevated cAMP levels suppress growth in C6 cells (a model of glioma) through treatment with forskolin, an AC activator, or a range of small molecule PDE inhibitors with differing selectivity profiles. Forskolin suppressed cell growth in a protein kinase A (PKA)-dependent manner by inducing a G2/M phase cell cycle arrest. In contrast, trequinsin (a non-selective PDE2/3/7 inhibitor), not only inhibited cell growth via PKA, but also stimulated (independent of PKA) caspase-3/-7 and induced an aneuploidy phenotype. Interestingly, a cocktail of individual PDE 2,3,7 inhibitors suppressed cell growth in a manner analogous to forskolin but not trequinsin. Finally, we demonstrate that concomitant targeting of both AC and PDEs synergistically elevated intracellular cAMP levels thereby potentiating their antiproliferative actions.

A predictive computational model reveals that GIV/girdin serves as a tunable valve for EGFR-stimulated cyclic AMP signals

Cellular levels of the versatile second messenger cyclic (c)AMP are regulated by the antagonistic actions of the canonical G protein → adenylyl cyclase pathway that is initiated by G-protein–coupled receptors (GPCRs) and attenuated by phosphodiesterases (PDEs). Dysregulated cAMP signaling drives many diseases; for example, its low levels facilitate numerous sinister properties of cancer cells. Recently, an alternative paradigm for cAMP signaling has emerged in which growth factor–receptor tyrosine kinases (RTKs; e.g., EGFR) access and modulate G proteins via a cytosolic guanine-nucleotide exchange modulator (GEM), GIV/girdin; dysregulation of this pathway is frequently encountered in cancers. In this study, we present a network-based compartmental model for the paradigm of GEM-facilitated cross-talk between RTKs and G proteins and how that impacts cellular cAMP. Our model predicts that cross-talk between GIV, G αs, and G αi proteins dampens ligand-stimulated cAMP dynamics. This prediction was experimentally verified by measuring cAMP levels in cells under different conditions. We further predict that the direct proportionality of cAMP concentration as a function of receptor number and the inverse proportionality of cAMP concentration as a function of PDE concentration are both altered by GIV levels. Taking these results together, our model reveals that GIV acts as a tunable control valve that regulates cAMP flux after growth factor stimulation. For a given stimulus, when GIV levels are high, cAMP levels are low, and vice versa. In doing so, GIV modulates cAMP via mechanisms distinct from the two most often targeted classes of cAMP modulators, GPCRs and PDEs.