Forskolin Research Articles

E3 ligase MDM2 mediates urea transporter-A1 ubiquitination under either constitutive or stimulatory conditions.

Posttranslational modifications are essential for the regulation of urea transporter-A1 (UT-A1), among which ubiquitination is a rather attractive and complex issue. Previously, our group reported that murine double minute 2 (MDM2) is one of the E3 ubiquitin ligases for UT-A1, and, later, we showed that ubiquitination contributes to the subcellular trafficking and stability of UT-A1. In the present study, we discovered that MDM2 interacts with UT-A1 in an AP50 (a component of the clathrin-coated pit)-dependent manner. However, their binding is irrelevant to the phosphorylatory status of UT-A1. Next, our findings indicated that MDM2 decreases the stability of either total or membrane UT-A1. On the cell membrane, MDM2 and ubiquitinated UT-A1 are both distributed in the lipid raft domain, and their linkage is obviously enhanced under forskolin (FSK) stimulation. In line with these results, in the diabetic rat, not only MDM2 but also ubiquitinated UT-A1 are intensified. Also, in vitro high glucose and angiotensin II play similar roles as FSK does on the association of MDM2 with UT-A1. In conclusion, MDM2 binds with UT-A1 and mediates its ubiquitination and degradation in an AP50-dependent manner, and their binding capacity is strengthened under FSK and diabetic milieu.

Fear renewal activates cyclic adenosine monophosphate signaling in the dentate gyrus.

BackgroundFear renewal, the context‐specific relapse of a conditioned fear after extinction, is a widely pursued model of post‐traumatic stress disorder and phobias. However, its cellular and molecular mechanisms remain poorly understood. The dentate gyrus (DG) has emerged as a critical locus of plasticity with relevance to memory, anxiety disorders, and depression, and it contributes to fear memory retrieval. Here, we have identified the role of the DG in fear renewal and its molecular mechanism.Materials and MethodsMuscimol (MUS), activator of cyclic adenosine monophosphate (cAMP) forskolin (FSK), inhibitor of protein kinase A (PKA), Rip‐cAMP, and a phosphodiesterase inhibitor rolipram were infused into DG of standard deviation rats before renewal testing. cAMP levels after fear renewal was measured by enzyme‐linked immunosorbent assay. The protein levels of phosphodiesterase 4 (PDE4) isoforms were tested by western blot. At last, the roles of cAMP signaling were also tested in the acquisition of fear conditioning, fear retrieval, and extinction.ResultsIntra‐DG treatment of MUS and Rp‐cAMP impaired fear renewal. FSK and rolipram exhibited the opposite effect, which also occurred in the retrieval of original fear memory. This change in fear renewal was regulated by PDE4 isoforms PDE4A, PDE4A5, and PDE4D. In addition, FSK and rolipram facilitated the acquisition of fear conditioning in long‐term memory, but not short‐term memory, while Rp‐cAMP impaired long‐term memory. For extinction, FSK and rolipram inhibited extinction process, while Rp‐cAMP facilitated fear extinction.ConclusionThese findings demonstrated that fear renewal activated cAMP signaling in the DG through decreased PDE4 activity. Because of the role of cAMP signaling in the acquisition or retrieval of fear conditioning and encoding of extinction, it is speculated that initial learning and extinction may have similarities in molecular mechanism, especially fear retrieval and fear renewal may share cAMP signaling pathway in the DG.

Interplay of transcriptional signaling by progesterone, cyclic AMP, and inflammation in myometrial cells: implications for the control of human parturition.

Parturition involves cellular signaling changes driven by the complex interplay between progesterone (P4), inflammation, and the cyclic adenosine monophosphate (cAMP) pathway. To characterize this interplay, we performed comprehensive transcriptomic studies utilizing eight treatment combinations on myometrial cell lines and tissue samples from pregnant women. We performed genome-wide RNA-sequencing on the hTERT-HM${}^{A/B}$ cell line treated with all combinations of P4, forskolin (FSK) (induces cAMP), and interleukin-1$\beta$ (IL-1$\beta$). We then performed gene set enrichment and regulatory network analyses to identify pathways commonly, differentially, or synergistically regulated by these treatments. Finally, we used tissue similarity index (TSI) to characterize the correspondence between cell lines and tissue phenotypes. We observed that in addition to their individual anti-inflammatory effects, P4 and cAMP synergistically blocked specific inflammatory pathways/regulators including STAT3/6, CEBPA/B, and OCT1/7, but not NF$\kappa$B. TSI analysis indicated that FSK + P4- and IL-1$\beta$-treated cells exhibit transcriptional signatures highly similar to non-laboring and laboring term myometrium, respectively. Our results identify potential therapeutic targets to prevent preterm birth and show that the hTERT-HM${}^{A/B}$ cell line provides an accurate transcriptional model for term myometrial tissue.

Inhibition by fluoxetine of LH-stimulated cyclic AMP synthesis in tumor Leydig cells partly involves AMPK activation.

Fluoxetine (FLX), a widely used antidepressant primarily acting as a selective serotonin reuptake inhibitor (SSRI), has been shown to exhibit other mechanisms of action in various cell types. Consequently, it might have unexpected adverse effects not related to its intended use, possibly in the endocrine regulation of reproduction. We show in the present report that after a 1-hour preincubation of MLTC-1 Leydig cells with FLX, the intracellular cyclic adenosine monophosphate (cAMP) responses to Luteinizing Hormone (LH) and forskolin (FSK) are reduced through AMPK-dependent and -independent pathways respectively. FLX at low concentrations (12.5μM and 25μM) induced this inhibition without triggering AMPK phosphorylation, while higher FLX concentrations (50μM and 100μM) induced AMPK phosphorylation and lowered ATP concentration similarly to Metformin. Pretreatment with the specific AMPK inhibitor Compound C (CpdC), significantly diminished the inhibition of cAMP synthesis caused by high concentration of FLX. Moreover, as expected FLX also caused a decline of steroidogenesis which is under the control of cAMP. Taken together, these findings demonstrate that the inhibition of cAMP synthesis by FLX is dose-dependent and occurs in MLTC-1 cells through two mechanisms, AMPK-independent and AMPK-dependent, at low and high concentrations, respectively. FLX also inhibited hormone-induced steroidogenesis in MLTC-1 cells and mouse testicular Leydig cells, suggesting similar mechanisms in both cell types.

Activation of the Calcium Sensing Receptor Decreases Secretagogue-Induced Fluid Secretion in the Rat Small Intestine.

BackgroundThe calcium-sensing receptor (CaSR) has been localized and characterized in numerous tissues throughout the body. In the mammalian gastrointestinal tract, the CaSR is known to act as a nutrient sensor and has recently been found to play a role in intestinal fluid and electrolyte balance. This study aims to demonstrate the functionality of the CaSR as a modulator of fluid secretion and absorption along the small intestine.MethodsSmall intestine regions (proximal, middle, and distal) were isolated from Sprague Dawley rats and loaded into an ex vivo intestinal perfusion device that provides independent intraluminal and extraluminal (serosa/basolateral) perfusion. The regions were perfused with 5 and 7 mM of Ca2+, both in the presence and absence of forskolin (FSK), a potent secretagogue. Control experiments were conducted with intraluminal perfusate containing standard Ringer-HEPES buffer with a physiological concentration of Ca2+ (1 mM). A second set of comparison experiments was performed with intraluminal perfusates containing AC-265347, a CaSR activator and agonist, in the presence of FSK. In all experimental conditions, the intraluminal perfusate contained fluorescein isothiocyanate (FITC)-inulin, a nonabsorbable fluorescent marker of secretion and/or absorption. Intraluminal fluorescence signal was utilized as a measure of water movement at the start of the experiment and every 15 min for 90 min.ResultsUnder physiological conditions, increasing the concentration of Ca2+ in the luminal perfusate reduced intestinal fluid secretion in all regions. At a Ca2+ concentration of 7 mM, net fluid absorption was observed in all regions. In the presence of FSK, 5 mM Ca2+ significantly decreased fluid secretion and 7 mM Ca2+ abolished FSK-induced fluid secretion. Intraluminal perfusion with 5 mM Ca2+ was as effective as AC-265347, in reducing secretagogue-induced fluid hypersecretion in the proximal and middle regions.ConclusionThis study concludes that apical CaSR is active along the small intestine. Its activation by Ca2+ and/or calcimimetics reduces fluid secretion in a dose-dependent manner, with higher Ca2+ concentrations, or application of a calcimimetic, leading to fluid absorption. We furthermore show that, in the presence of FSK, receptor activation abates FSK secretagogue-induced fluid secretion. This presents a new therapeutic target to address secretory diarrheal illnesses.

Specificity of NHERF1 regulation of GPCR signaling and function in human airway smooth muscle.

Na+/H+ exchanger regulatory factor 1 (NHERF1; also known as ezrin-radixin-moesin-binding phosphoprotein 50) is a PSD-95, disc large, zona occludens-1 adapter that acts as a scaffold for signaling complexes and cytoskeletal-plasma membrane interactions. NHERF1 is crucial to β-2-adrenoceptor (β2AR)-mediated activation of cystic fibrosis transmembrane conductance regulator (CFTR) in epithelial cells, and NHERF1 has been proposed to mediate the recycling of internalized β2AR back to the cell membrane. In the current study, we assessed the role of NHERF1 in regulating cAMP-mediated signaling and immunomodulatory functions in airway smooth muscle (ASM). NHERF1 knockdown attenuated the induction of (protein kinase A) phospho-vasodilator-stimulated phosphoprotein (p-VASP) by isoproterenol (ISO), prostaglandin E2 (PGE2), or forskolin (FSK) as well as the induction of p-heat shock protein 20 after 4 h of stimulation with ISO and FSK. NHERF1 knockdown fully abrogated the ISO-, PGE2-, and FSK-induced IL-6 gene expression and cytokine production without affecting cAMP-mediated phosphodiesterase 4D (PDE4D) gene expression, phospho-cAMP response element-binding protein (p-CREB), and cAMP response element (CRE)-Luc, or PDGF-induced cyclin D1 expression. Interestingly, NHERF1 knockdown prevented ISO-induced chromatin-binding of the transcription factor CCAAT-enhancer-binding protein-β (c/EBPβ). c/EBPβ knockdown almost completely abrogated the cAMP-mediated IL-6 but not PDE4D gene expression. The differential regulation of cAMP-induced signaling and gene expression in our study indicates a role for NHERF1 in the compartmentalization of cAMP signaling in ASM.-Pera, T., Tompkins, E., Katz, M., Wang, B., Deshpande, D. A., Weinman, E. J., Penn, R. B. Specificity of NHERF1 regulation of GPCR signaling and function in human airway smooth muscle.

Modification of the first constant domain of heavy chain enabled effective folding of functional anti-forskolin antigen-binding fragment for sensitive quantitative analysis.

The assembly between heavy and light chains is a critical step of immunoglobulin (Ig) and fragment antigen-binding (Fab) antibody expression and of their binding activity. The genes encoding Fab were obtained from hybridoma cells secreting monoclonal antibody (MAb, IgG2b) against adenylate cyclase activator forskolin (FOR). The subclass of the first constant domain of heavy chain (CH 1) of IgG2b was modified to IgG1 via overlap extension polymerase chain reaction and expressed via Escherichia coli bacterial system. Since both Fabs (IgG2b and IgG1) were expressed as inclusion bodies, functional analysis was performed after in vitro refolding via stepwise dialysis. The result indicated that the folding efficiency between VH -CH 1 and VL -CL was improved by the CH 1 modification from IgG2b to IgG1 subclass, although their specificity for FOR was not altered. Effective folding of IgG1 was also observed when they were expressed in the hemolymph of silkworm larvae using the Bombyx mori nuclear polyhedrosis virus bacmid system. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was then developed for the determination of FOR using effectively prepared Fab IgG1. The sensitivity of FOR determination was in the range of 3.91-62.5 ng/mL with less than 9% relative standard deviation, implying the sensitive and reliable analysis of developed icELISA. In addition, high accuracy of the icELISA was supported by the results of spiked-and-recovery tests, ranging from 100.2 to 102.3%. Therefore, Fab could be utilized reliably for icELISA instead of the more expensive MAb. Collectively, this approach improved productivity of Fab and reduced the cost of antibody production.

GLI inhibitors overcome Erlotinib resistance in human pancreatic cancer cells by modulating E-cadherin

Inhibition of hedgehog (Hh) signalling pathway, including its end effector GLI1, can reverse epithelial-to-mesenchymal transition (EMT) which plays an important role in drug resistance of pancreatic cancer cells to Erlotinib (ETB). This study investigated the effect of GLI inhibitors Forskolin (FSK), GANT-61 (GNT), and Arsenic trioxide (ATX) on suppressing the resistance of pancreatic cancer cells to ETB. The effect of GLI inhibitors was evaluated by measuring mRNA expression levels of EMT factors using quantitative RT-PCR. Immunocytochemistry and flow cytometry were used to assess E-cadherin (E-Cad) and GLI1 protein levels. MTT and apoptosis assays were used to evaluate the synergistic effects for the combination treatment of each GLI inhibitor with ETB. Pancreatic cancer cells PANC-1 treated by GNT showed the highest significant reduction in mRNA levels of GLI1 and other EMT pathway genes. Moreover, GNT was able to upregulate E-Cad and downregulate GLI1 proteins, more than FSK, while ATX had no effect. Apoptosis levels of PANC-1 cells following treatment with LD30 concentrations of FSK, GNT, or ATX, showed 57%, 62% and 67%, respectively, in comparison to ETB (∼48%). Importantly, combination treatments of ETB with either FSK, GNT, or ATX demonstrated a significant increase in apoptotic cells reaching 61% (ETB + FSK), 80% (ETB + GNT) or 88% (ETB + ATX). FSK did not have much effect on the drug resistance of PANC-1 cells to ETB. However, GNT, but more effectively ATX, were able to reduce the drug resistance of this cell line to ETB.

B-cell translocation gene 2 enhances fibroblast growth factor 21 production by inducing Kruppel-like factor 15

Fibroblast growth factor 21 (FGF21) is a hormone that is vital for the regulation of metabolic homeostasis. In the present study, we report that Kruppel-like factor 15 (KLF15) is a novel mediator of b-cell translocation gene 2 (BTG2)-induced FGF21 biosynthesis. The expression levels of hepatic Fgf21, Btg2, and Klf15, and the production of serum FGF21 increased significantly in fasted and forskolin (FSK)-treated mice. The overexpression of Btg2 using an adenoviral delivery system elevated FGF21 production by upregulating Klf15 transcription. Interaction studies indicated that BTG2 was co-immunoprecipitated with KLF15 and recruited by the Fgf21 promoter. The disruption of hepatic Btg2 and Klf15 genes markedly attenuated the induction of Fgf21 expression and FGF21 biosynthesis in fasted mice. Similarly, the FSK-mediated induction of Fgf21 promoter activity was strikingly ablated by silencing of Btg2 and Klf15. Taken together, these findings suggest that KLF15 and BTG2 are mediators of fasting-induced hepatic FGF21 expression. Therefore, targeting BTG2 and KLF15 might be a therapeutically important strategy for combat metabolic dysfunction.

125 Iodide as a surrogate tracer for epithelial chloride transport by the mouse large intestine in vitro.

What is the central question of this study? The tracer 36 Cl- , currently used to measure transepithelial Cl- fluxes, has become prohibitively expensive, threatening its future use. 125 Iodide, previously validated alongside 36 Cl- as a tracer of Cl- efflux by cells, has not been tested as a surrogate for 36 Cl- across epithelia. What is the main finding and its importance? We demonstrate that 125 I- can serve as an inexpensive replacement for measuring Cl- transport across mouse large intestine, tracking Cl- transport in response to cAMP stimulation (inducing Cl- secretion) in the presence and absence of the main gastrointestinal Cl- -HCO3- exchanger, DRA. Chloride transport is important for driving fluid secretion and absorption by the large intestine, with dysregulation resulting in diarrhoea-associated pathologies. The radioisotope 36 Cl- has long been used as a tracer to measure epithelial Cl- transport but is prohibitively expensive. 125 Iodide has been used as an alternative to 36 Cl- in some transport assays but has never been validated as an alternative for tracing bidirectional transepithelial Cl- fluxes. The goal of this study was to validate 125 I- as an alternative to 36 Cl- for measurement of Cl- transport by the intestine. Simultaneous fluxes of 36 Cl- and 125 I- were measured across the mouse caecum and distal colon. Net Cl- secretion was induced by the stimulation of cAMP with a cocktail of forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX). Unidirectional fluxes of 125 I- correlated well with 36 Cl- fluxes after cAMP-induced net Cl- secretion, occurring predominantly through a reduction in the absorptive mucosal-to-serosal Cl- flux rather than by stimulation of the secretory serosal-to-mucosal Cl- flux. Correlations between 125 I- fluxes and 36 Cl- fluxes were maintained in epithelia from mice lacking DRA (Slc26a3), the main Cl- -HCO3- exchanger responsible for Cl- absorption by the large intestine. Lower rates of Cl- and I- absorption in the DRA knockout intestine suggest that DRA might have a previously unrecognized role in iodide uptake. This study validates that 125 I- traces transepithelial Cl- fluxes across the mouse large intestine, provides insights into the mechanism of net Cl- secretion and suggests that DRA might be involved in intestinal iodide absorption.

Isoforskolin and forskolin attenuate lipopolysaccharide-induced inflammation through TLR4/MyD88/NF-κB cascades in human mononuclear leukocytes.

The principal active component of isoforskolin (ISOF) is from the plant Coleus forskohlii, native to China, which has attracted much attention for its biological effects. We hypothesize that ISOF and forskolin (FSK) pretreatment attenuates inflammation induced by lipopolysaccharide (LPS) related to toll‐like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and nuclear factor kappa B (NF‐κB) signaling. Mononuclear leukocytes (MLs) from healthy donors' blood samples were separated by using density gradient centrifugation. Protein levels of TLR4, MyD88, and NF‐κB were detected using western blot and inflammatory cytokines interleukin (IL) 1β, IL‐2, IL‐6, IL‐21, IL‐23, tumor necrosis factor (TNF) α, and TNF‐β were tested by enzyme‐linked immunosorbent assay and Quantibody array in MLs. Our results showed that LPS augmented the protein levels of TLR4, MyD88, and NF‐κB in MLs and the production of IL‐1β, IL‐2, IL‐6, IL‐21, IL‐23, TNF‐α, and TNF‐β in supernatants of MLs. Despite treatment with ISOF and FSK prior to LPS, the protein levels of TLR4, MyD88, NF‐κB, IL‐1β, IL‐2, IL‐6, IL‐21, IL‐23, TNF‐α, and TNF‐β in MLs were apparently decreased. roflumilast (RF) and dexamethasone (DM) had a similar effect on MLs with ISOF and FSK. Our results, for the first time, have shown that ISOF and FSK attenuate inflammation in MLs induced by LPS through down‐regulating protein levels of IL‐1β and TNF‐α, in which TLR4/MyD88/NF‐κB signal pathway could be involved.

Anticataract activity of forskolin by inhibiting polyol pathway for the prevention of diabetic complication

Pharmacognosy Research,2019,11,4,352-355.DOI:10.4103/pr.pr_68_19Published:November,2019Type:Original ArticleAuthors:Damera Sujatha, Ajmera Rama Rao, and Ciddi Veeresham Author(s) affiliations:Damera Sujatha, Ajmera Rama Rao, Ciddi Veeresham Department of Pharmacognosy, University College of Pharmaceutical Sciences, Kakatiya University, Warangal, Telangana, India. Abstract:Background: Cataract is the opacification or optical dysfunction of the crystalline lens. Diabetes has been considered to be one of the major risk factors of cataract. Objective: The present study was designed to evaluate the anticataract activity of antioxidants such as Coleus forskohlii (CF) Briq and its phytoconstituent forskolin (FS) which were subjected to prevent cataract formation in vitro on glucose‑induced cataract model. Materials and Methods: Goat lenses were incubated in Krebs‑Ringer bicarbonate buffer pH 7.5 (supplemented with Taxim and streptomycin) containing 55 mM glucose (cataractogenesis) with fidarestat; CF methanolic extract; and FS at a concentration of 1 µg/mL, 100 µg/mL, and 10 µg/mL for 24 hr at 37°C with 5% CO2 and 95% air. Glucose‑induced opacification of goat lens began 8–10 hr after incubation and was complete in 24 hr. Polyol (galactitol) levels in incubated lenses were estimated spectrophotometrically. Results: Cataractous lenses showed higher content of galactitol. However, lens treated with fidarestat, CF methanolic extract, and its phytoconstituent FS showed lower content of galactitol. Conclusion: CF and FS prevented the formation and progress of cataract by glucose, as evidenced by lens transparencies with photographic evaluation and lens galactitol levels. Keywords:Aldose reductase, Cataract, Coleus forskohlii, Forskolin, Polyol pathway.View:PDF (941.95 KB)

Forskolin inhibited the secretion of interleukin-1β in activated human THP-1 macrophages

Objective To investigate the effects of Forskolin on the activation of nucleotide-binding oligomerization domain like receptor family, pyrin domain-containing 3(NLRP-3) inflammasome and the secretion of interleukin-1β(IL-1β) in activated human THP-1 macrophages, which can provide evidence for clinical treatment of inflammatory diseases in children. Methods Human monocyte cell line THP-1 cells were induced to differentiate into macrophages by Phorbol-12-myristate-13-acetate(PMA), and Forskolin(10, 50, 100 μmol/L) stimulated activated macrophages by nigericin.The mRNA of the inflammasome markers NLRP-3, IL-1β and Caspase-1 were detected by real-time quantitative polymerase chain reaction(qPCR) method.The protein of NLRP-3, pro-IL-1β, pro-Caspase-1 and Caspase-1 were detected by Western blot.The secretion of inflammatory cytokines IL-1β was detected by enzyme-linked immuno-sorbent assay(ELISA). Results Nigericin activated the cells and the mRNA expression of NLRP-3, IL-1β and Caspase-1 increased by 7.59 times(P 0.05). Western blot showed that Forskolin also had no effect on the protein expression of pro-Caspase-1 and pro-IL-1β in activated THP-1 macrophages(P>0.05), but the expression of NLRP-3 and Caspase-1 decreased significantly (P<0.001). The results of ELISA showed that the IL-1β secretion increased from basal 584.0 nmol/L to activated 2 695.6 nmol/L(t=16.031 1, P<0.001) on THP-1 macrophages by nigericin, but Forskolin(10, 50, 100 μmol/L) reduced it to 1 858.4 nmol/L(t=5.365 5, P<0.001), 1 467.9 nmol/L(t=8.047 5, P<0.001) and 1 246.7 nmol/L(t=10.199 0, P<0.001) on the activated THP-1 macrophages. Conclusions Forskolin can not affect the expression of pro-cytokines at the gene transcription and protein translation levels in activated THP-1 cells, but inhibit the secretion of inflammatory cytokines IL-1β, so as to inhibit inflammatory response, which can be used to treat pediatric inflammatory diseases caused by IL-1β. Key words: Inflammation; Forskolin; Macrophage; Inflammasome; Interleukin-1β

Cold exposure induces nuclear translocation of CRTC3 in brown adipose tissue.

In mammals, cold stress activates the cAMP-protein kinase A (PKA) signaling pathway, increases brown adipose tissue (BAT) activity, and induces thermogenesis to maintain body temperature. The cAMP responsive element binding protein (CREB)-regulated transcription coactivator 3 (CRTC3) plays important role in adipose development and energy metabolism. However, the effect of cold exposure on the intracellular localization of CRTC3 in BAT is unclear. Here, we report that cold-treated mice have higher expression of uncoupling protein 1 (UCP1) in adipose tissues and lower body weights and fat masses. Notably, cold exposure results in the nuclear translocation of CRTC3 in BAT. Moreover, forskolin (FSK), the activator of PKA pathway, induces the nuclear translocation of CRTC3 in brown adipocytes. At the molecular level, cold exposure and FSK treatment decrease liver kinase B1 (Lkb1) expression in brown adipocytes, which is related to the nuclear localization of CRTC3. These results demonstrate that the localization of CRTC3 involves in regulating cold-induced upregulation of UCP1 in BAT and provide useful information for understanding the molecular regulation of BAT thermogenesis induced by a cold environment.

GLP-1 attenuates Ang II-induced proliferation and migration in rat aorta smooth muscle cells via inhibition of the RhoA/ROCK2 signaling pathway.

Glucagon-like peptide 1 (GLP-1), a neuroendocrine hormone produced by the gastrointestinal tract, plays a significant role in blood glucose regulation; drugs derived from GLP-1 are currently used for the treatment of type 2 diabetes. In addition to regulating glucose homeostasis, the protective effects of GLP-1 on the cardiovascular system are also of interest. However, the vascular protective mechanisms of GLP-1 remain unclear. The present study was designed to evaluate the role of GLP-1 in the proliferation and migration of vascular smooth muscle cells, and the underlying mechanisms. In this study, proliferation, migration, cyclin D1 expression, and phosphorylation of MLC, as well as RhoA and Rho-associated coiled-coil forming protein kinase 2 (ROCK2) expression, were increased in rat aorta smooth muscle cells (RASMCs) following incubation with angiotensin II (Ang II). These effects were significantly attenuated by GLP-1, forskolin (a cAMP activator) and Y-27632 (a ROCK2 inhibitor). However, H89 (a PKA inhibitor) inhibited the action of GLP-1, both in terms of inhibition of RASMC proliferation and migration, and RHOA/ROCK2 expression. These results indicate that GLP-1 inhibits Ang II-induced RASMC proliferation and migration via the cAMP/PKA/RhoA/ROCK2 signaling pathway. Our data suggest that GLP-1 should be considered for use in the clinical treatment of cardiovascular diseases, in addition to its current use in the treatment of diabetes mellitus.

Identification of intracellular peptides associated with thermogenesis in human brown adipocytes.

Currently, brown adipose tissue (BAT) is a therapeutic target in obesity and diabetes, but the mechanism of BAT activation remains unclear. Because increasing emphasis has been placed on the role of intracellular peptides in biological processes, we conducted a study to gain insight into the mechanism of BAT activation by using a peptidomic approach and then attempted to identify peptides that are capable of activating BAT. In the presentstudy, we generated the peptidomic profile of the intracellular peptides in brown adipocytes treated with forskolin (FSK) using a peptidomic approach. Then, the differentially expressed peptides were evaluated via Gene Ontology (GO) enrichment, KEGG pathway, and protein-protein interaction (PPI) network analysis. Finally, we selected candidate peptides for further validation via assessing the expression levels of UCP-1 and PGC-1α in brown adipocytes exposed to the peptides. A total of 4,370 peptides were identified, of which 951 were upregulated and 379 were downregulated after FSK treatment. Bioinformatic analysis demonstrated that the ECM-receptor interaction GO term was the most enriched and that collagen alpha-related proteins exhibited the highest degree of PPI. Four peptides separately derived from TSC22 domain family protein 1 (T22D1), bromodomain and WD repeat-containing protein 1 (BRWD1), protein piccolo (PCLO), and collagen alpha-1 (III) chain (CO3A1) increased the expression levels of UCP-1 and PGC-1α. ECM-receptor interaction may play an important role in the process of FSK-stimulated BAT activation, and the pT22D1tide, pBRWD1tide, pPCLOtide, and pCO3A1tide peptides potentially promote BAT thermogenesis.

Functional polarization of human hepatoma HepaRG cells in response to forskolin

HepaRG is an original human hepatoma cell line, acquiring highly differentiated hepatic features when exposed to dimethylsulfoxide (DMSO). To search alternatives to DMSO, which may exert some toxicity, we have analyzed the effects of forskolin (FSK), a cAMP-generating agent known to favor differentiation of various cell types. FSK used at 50 µM for 3 days was found to promote polarization of high density-plated HepaRG cells, i.e., it markedly enhanced the formation of functional biliary canaliculi structures. It also increased expressions of various hepatic markers, including those of cytochrome P-450 (CYP) 3A4, of drug transporters like NTCP, OATP2B1 and BSEP, and of metabolism enzymes like glucose 6-phosphatase. In addition, FSK-treated HepaRG cells displayed enhanced activities of CYP3A4, NTCP and OATPs when compared to untreated cells. These polarizing/differentiating effects of FSK were next shown to reflect not only the generation of cAMP, but also the activation of the xenobiotic sensing receptors PXR and FXR by FSK. Co-treatment of HepaRG cells by the cAMP analog Sp-5,6-DCl-cBIMPS and the reference PXR agonist rifampicin reproduced the polarizing effects of FSK. Therefore, FSK may be considered as a relevant alternative to DMSO for getting polarized and differentiated HepaRG cells, notably for pharmacological and toxicological studies.

Study on site-specific expression of bone formation and resorption factors in human dental follicles.

We sought to investigate site‐specific expression of bone‐regulatory factors expressed by human dental follicles and to compare the stimulated expression of tumour necrosis factor (ligand) superfamily, member 11/tumour necrosis factor receptor superfamily, member 11b (RANKL/OPG) in human dental follicle cells (HDFCs) from different patients. Analysis of bone‐regulatory markers in follicles from 12 different study participants was performed using RT‐qPCR and immunofluorescence; apical and coronal segments from each dental follicle were processed independently. Four additional dental follicles were used for cell cultures; HDFCs were precultured in osteogenic medium to initiate differentiation and thereafter cultured with 10−6 M forskolin (FSK) to activate the protein kinase cAMP (PKA/cAMP) signalling pathway and induce RANKL/OPG expression. We demonstrate that RANKL expression is significantly higher in the coronal part of follicles than in the apical part. High levels of collagen type 1 (COL1), alkaline phosphatase (ALP) and Gap‐junction protein, alpha 1, 43 kDa (CX43) were expressed, whereas expression of Sp7 transcription factor (OSX), bone morphogenetic protein 2 (BMP2), colony‐stimulating factor 1 (CSF‐1), chemokine (C‐C motif) ligand 2 (MCP1), and OPG was low in all samples. The immunofluorescence localization of CSF‐1, MCP1, osteocalcin (OCN), RANKL, and BMP2 was not specific for either part of the follicles. In conclusion, a consistently high expression of CX43 suggests that gap‐junction communication in HDFCs is essential for the eruption process. Furthermore, the induced expression of RANKL in HDFCs varies significantly between individuals and may relate to clinical variations in tooth eruption.

Role of Cyclic Adenosine Monophosphate in Myopic Scleral Remodeling in Guinea Pigs: A Microarray Analysis.

Myopia induction accompanies increased scleral cyclic adenosine phosphate (cAMP) levels and collagen degradation in mammalian models. We compared the scleral gene expression changes following monocular form deprivation (FD) with those induced by adenylate cyclase activation with forskolin (FSK) in guinea pigs. Guinea pigs were assigned to FD, FSK-treated, and age-matched (AM) control groups. FSK was injected monocularly into the inferior palpebral subconjunctiva daily for 4 days. After scleral RNA extraction, a gene microarray scanner and software were used to evaluate the gene expression patterns, followed by pathway analysis using Gene Ontology tools. Quantitative PCR (qPCR) was used to analyze the expression of 10 candidate genes in separate sets of form-deprived, vehicle-injected, and AM animals. FSK injections differentially regulated 13 collagen subtypes compared to AM and FD groups. FSK also downregulated Acta2 and Tgf-β2 compared to the AM eyes. Collagen subtypes and Acta2 underwent larger downregulation in the FSK group than during FD. FSK differentially regulated Rarb, Rxrg, Fzd5, Ctnnd2, Dkk2, and Dkk3, which have been linked to ocular growth. Only a few genes were differentially expressed between the FD and AM groups. There was 80% agreement in the direction of gene regulation between microarray and qPCR results. No significant differences were identified between vehicle-injected and AM eyes. Collagen, a major scleral extracellular matrix component, is degraded during myopia. Given that FSK and FD both promote myopia through increased collagen degradation, targeting cAMP signaling pathway genes could suppress myopia development.

CFTR prevents neuronal apoptosis following cerebral ischemia reperfusion via regulating mitochondrial oxidative stress.

The cystic fibrosis transmembrane conductance regulator (CFTR) is linked to cell apoptosis and abundantly expressed in brain tissue. Mitochondrial oxidative stress plays a key role in activating apoptotic pathway following cerebral ischemia reperfusion (IR) injury. Reduced glutathione (GSH) is exclusively synthesized in cytosol but distributed in mitochondria. In the present study, we investigated whether CFTR affected mitochondrial oxidative stress via regulating GSH and thereby protected neurons against apoptosis following cerebral IR. Brains were subjected to global IR by four-vessel occlusion and CFTR activator forskolin (FSK) was used in vivo. CFTR silence was performed in vitro for neurons by RNA interference. We found that FSK suppressed neuronal apoptosis whereas CFTR silence enhanced neuronal apoptosis. FSK prevented the elevations in reactive oxygen species (ROS) and caspase activities while FSK inhibited the reductions in complex I activity and mitochondrial GSH level following IR. FSK decreased mitochondrial oxidative stress partially and preserved mitochondrial function. On the contrary, CFTR silence exaggerated mitochondrial dysfunction. CFTR loss increased hydrogen peroxide (H2O2) level and decreased GSH level in mitochondria. Importantly, we showed that CFTR was located on mitochondrial membrane. GSH transport assay suggested that GSH decrease may be a consequence not a reason for mitochondrial oxidative stress mediated by CFTR disruption. Our results highlight the central role of CFTR in the pathogenesis of cerebral IR injury. CFTR regulates neuronal apoptosis following cerebral IR via mitochondrial oxidative stress-dependent pathway. The mechanism of CFTR-mediated mitochondrial oxidative stress needs further studies. KEY MESSAGES: CFTR activation protects brain tissue against IR-induced apoptosis and oxidative stress. CFTR disruption enhances H2O2-induced neuronal apoptosis and CFTR loss leads to mitochondrial oxidative stress. CFTR regulates IR-induced neuronal apoptosis via mitochondrial oxidative stress. CFTR may be a potential therapeutic target to cerebral IR damage.