Foxa2 and Pdx1 cooperatively regulate postnatal maturation of pancreatic ß-cells.

OBJECTIVE: The transcription factors (TF) Foxa2 and Pdx1 are key regulators of beta-cell (ß-cell) development and function. Mutations of these TFs or their respective cis-regulatory consensus binding sites have been linked to maturity diabetes of the young (MODY), pancreas agenesis, or diabetes susceptibility in human. Although Foxa2 has been shown to directly regulate Pdx1 expression during mouse embryonic development, the impact of this gene regulatory interaction on postnatal ß-cell maturation remains obscure. METHODS: In order to easily monitor the expression domains of Foxa2 and Pdx1 and analyze their functional interconnection, we generated a novel double knock-in homozygous (FVFPBFDHom) fluorescent reporter mouse model by crossing the previously described Foxa2-Venus fusion (FVF) with the newly generated Pdx1-BFP (blue fluorescent protein) fusion (PBF) mice. RESULTS: Although adult PBF homozygous animals exhibited a reduction in expression levels of Pdx1, they are normoglycemic. On the contrary, despite normal pancreas and endocrine development, the FVFPBFDHom reporter male animals developed hyperglycemia at weaning age and displayed a reduction in Pdx1 levels in islets, which coincided with alterations in ß-cell number and islet architecture. The failure to establish mature ß-cells resulted in loss of ß-cell identity and trans-differentiation towards other endocrine cell fates. Further analysis suggested that Foxa2 and Pdx1 genetically and functionally cooperate to regulate maturation of adult ß-cells. CONCLUSIONS: Our data show that the maturation of pancreatic ß-cells requires the cooperative function of Foxa2 and Pdx1. Understanding the postnatal gene regulatory network of ß-cell maturation will help to decipher pathomechanisms of diabetes and identify triggers to regenerate dedifferentiated ß-cell mass.

Impact of islet architecture on ß-cell heterogeneity, plasticity and function.

Although ß-cell heterogeneity was discovered more than 50 years ago, the underlying principles have been explored only during the past decade. Islet-cell heterogeneity arises during pancreatic development and might reflect the existence of distinct populations of progenitor cells and the developmental pathways of endocrine cells. Heterogeneity can also be acquired in the postnatal period owing to ß-cell plasticity or changes in islet architecture. Furthermore, ß-cell neogenesis, replication and dedifferentiation represent alternative sources of ß-cell heterogeneity. In addition to a physiological role, ß-cell heterogeneity influences the development of diabetes mellitus and its response to treatment. Identifying phenotypic and functional markers to discriminate distinct ß-cell subpopulations and the mechanisms underpinning their regulation is warranted to advance current knowledge of ß-cell function and to design novel regenerative strategies that target subpopulations of ß cells. In this context, the Wnt/planar cell polarity (PCP) effector molecule Flattop can distinguish two unique ß-cell subpopulations with specific transcriptional signatures, functional properties and differential responses to environmental stimuli. In vivo targeting of these ß-cell subpopulations might, therefore, represent an alternative strategy for the future treatment of diabetes mellitus.

Identification of proliferative and mature ß-cells in the islets of Langerhans.

Insulin-dependent diabetes is a complex multifactorial disorder characterized by loss or dysfunction of ß-cells. Pancreatic ß-cells differ in size, glucose responsiveness, insulin secretion and precursor cell potential; understanding the mechanisms that underlie this functional heterogeneity might make it possible to develop new regenerative approaches. Here we show that Fltp (also known as Flattop and Cfap126), a Wnt/planar cell polarity (PCP) effector and reporter gene acts as a marker gene that subdivides endocrine cells into two subpopulations and distinguishes proliferation-competent from mature ß-cells with distinct molecular, physiological and ultrastructural features. Genetic lineage tracing revealed that endocrine subpopulations from Fltp-negative and -positive lineages react differently to physiological and pathological changes. The expression of Fltp increases when endocrine cells cluster together to form polarized and mature 3D islet mini-organs. We show that 3D architecture and Wnt/PCP ligands are sufficient to trigger ß-cell maturation. By contrast, the Wnt/PCP effector Fltp is not necessary for ß-cell development, proliferation or maturation. We conclude that 3D architecture and Wnt/PCP signalling underlie functional ß-cell heterogeneity and induce ß-cell maturation. The identification of Fltp as a marker for endocrine subpopulations sheds light on the molecular underpinnings of islet cell heterogeneity and plasticity and might enable targeting of endocrine subpopulations for the regeneration of functional ß-cell mass in diabetic patients.

Targeting insulin-producing beta cells for regenerative therapy.

Pancreatic beta cells differ in terms of glucose responsiveness, insulin secretion and proliferative capacity; however, the molecular pathways that regulate this cellular heterogeneity are unknown. We have identified the Wnt-planar cell polarity (PCP) effector Flattop (FLTP) as a biomarker that identifies mature beta cells in the islets of Langerhans. Interestingly, three-dimensional architecture and Wnt-PCP ligands are sufficient to trigger mouse and human beta cell maturation. These results highlight the fact that novel biomarkers shed light on the long-standing mystery of beta cell heterogeneity and identify the Wnt-PCP pathway as triggering beta cell maturation. Understanding heterogeneity in the islets of Langerhans might allow targeting of beta cell subpopulations for regenerative therapy and provide building principles for stem cell-derived islets. This review summarises a presentation given at the 'Can we make a better beta cell?' symposium at the 2015 annual meeting of the EASD. It is accompanied by two other reviews on topics from this symposium (by Amin Ardestani and Kathrin Maedler, DOI: 10.1007/s00125-016-3892-9 , and by Harry Heimberg and colleagues, DOI: 10.1007/s00125-016-3879-6 ) and a commentary by the Session Chair, Shanta Persaud (DOI: 10.1007/s00125-016-3870-2 ).

Microalbuminuria: target for renoprotective therapy PRO.

Drug efficacy is ascertained using clinically meaningful outcomes that directly affect the well-being of patients. However, in studies of chronic kidney disease progression, clinically meaningful outcomes like end-stage renal disease take a long time to occur. The use of surrogate end points/markers as replacement for clinical outcomes is tempting as it may reduce sample size requirements, shorten follow-up time, facilitate trial conduct, and allow the performance of intervention trials in earlier stages of kidney disease to be carried out. We here reviewed recent data supporting the use of microalbuminuria as a valid surrogate end point in clinical trials of chronic kidney disease. We provide data that albuminuria is associated with worse renal prognosis and that pharmacological treatment aimed to reduce albuminuria levels delays the progression of renal disease and the occurrence of clinical outcomes. Furthermore, we review new studies showing that albumin is not only an inert molecule but also directly affects the function of several cell types in the kidney and may have a pathogenic role in renal disease. Accepting microalbuminuria as a surrogate marker for renal outcomes will lead to less resource-consuming hard outcome trials, will accelerate the development of drugs for chronic kidney disease, and enable earlier access of these drugs to individual patients.

The effect of RAAS blockade on the progression of diabetic nephropathy.

The renin-angiotensin-aldosterone system (RAAS) has a key role in the regulation of blood pressure, sodium and water balance, and cardiovascular and renal homeostasis. In diabetic nephropathy, excessive activation of the RAAS results in progressive renal damage. RAAS blockade using angiotensin-converting-enzyme inhibitors or angiotensin-receptor blockers is the cornerstone of treatment of diabetic renal disease. Alternative RAAS-blockade strategies include renin inhibition and aldosterone blockade. Data from small initial studies of these agents are promising. However, single-agent interventions do not fully block the RAAS and patients treated with these therapies remain at high residual renal risk. Approaches to optimize drug responses include dietary changes and increasing dosages. The theoretically attractive option of combining different RAAS interventions has also been tested in clinical trials but long-term outcomes were disappointing. However, dual RAAS blockade might represent a good therapeutic option for specific patients. A better knowledge of the pathophysiology of the RAAS is crucial to fully understand the mechanisms of action of RAAS blockers and to exploit their renoprotective effects. Moreover, lifestyle interventions or diagnostic tools might be used to optimize RAAS blockade and identify those patients who are most likely to benefit from the therapy.

Management of hyperkalaemia consequent to mineralocorticoid-receptor antagonist therapy.

Mineralocorticoid-receptor antagonists (MRAs) reduce blood pressure and albuminuria in patients treated with angiotensin-converting-enzyme inhibitors or angiotensin-II-receptor blockers. The use of MRAs, however, is limited by the occurrence of hyperkalaemia, which frequently occurs in patients older than 65 years with impaired kidney function, and/or diabetes. Patients with these characteristics might still benefit from MRA therapy, however, and should not be excluded from this treatment option. This limitation raises the question of how to optimize the therapeutic use of MRAs in this population of patients. Understanding the individual variability in patients' responses to MRAs, in terms of albuminuria, blood pressure and serum potassium levels, might lead to targeted intervention. MRA use might be restricted to patients with high levels of mineralocorticoid activity, evaluated by circulating renin and aldosterone levels or renal excretion of potassium. In addition, reviewing the patient's diet and concomitant medications might prove useful in reducing the risk of developing subsequent hyperkalaemia. If hyperkalaemia does develop, treatment options exist to decrease potassium levels, including administration of calcium gluconate, insulin, ß(2)-agonists, diuretics and cation-exchange resins. In combination with novel aldosterone blockers, these strategies might offer a rationale with which to optimize therapeutic intervention and extend the population of patients who can benefit from use of MRAs.

Anti-inflammatory role of the cAMP effectors Epac and PKA: implications in chronic obstructive pulmonary disease.

Cigarette smoke-induced release of pro-inflammatory cytokines including interleukin-8 (IL-8) from inflammatory as well as structural cells in the airways, including airway smooth muscle (ASM) cells, may contribute to the development of chronic obstructive pulmonary disease (COPD). Despite the wide use of pharmacological treatment aimed at increasing intracellular levels of the endogenous suppressor cyclic AMP (cAMP), little is known about its exact mechanism of action. We report here that next to the ß(2)-agonist fenoterol, direct and specific activation of either exchange protein directly activated by cAMP (Epac) or protein kinase A (PKA) reduced cigarette smoke extract (CSE)-induced IL-8 mRNA expression and protein release by human ASM cells. CSE-induced IκBα-degradation and p65 nuclear translocation, processes that were primarily reversed by Epac activation. Further, CSE increased extracellular signal-regulated kinase (ERK) phosphorylation, which was selectively reduced by PKA activation. CSE decreased Epac1 expression, but did not affect Epac2 and PKA expression. Importantly, Epac1 expression was also reduced in lung tissue from COPD patients. In conclusion, Epac and PKA decrease CSE-induced IL-8 release by human ASM cells via inhibition of NF-κB and ERK, respectively, pointing at these cAMP effectors as potential targets for anti-inflammatory therapy in COPD. However, cigarette smoke exposure may reduce anti-inflammatory effects of cAMP elevating agents via down-regulation of Epac1.

Protein kinase A and the exchange protein directly activated by cAMP (Epac) modulate phenotype plasticity in human airway smooth muscle.

BACKGROUND AND PURPOSE: Platelet-derived growth factor (PDGF) modulates the airway smooth muscle (ASM) 'contractile' phenotype to a more 'proliferative' phenotype, resulting in increased proliferation and reduced contractility. Such phenotypic modulation may contribute to airway remodelling in asthma. We have previously shown that the cAMP effector molecules, protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac) inhibited PDGF-induced phenotypic modulation in bovine ASM. Here, we investigated these mechanisms in human ASM strips and cells. EXPERIMENTAL APPROACH: ASM strips were incubated with PDGF in the absence or presence of the activators of Epac (8-pCPT-2'-O-Me-cAMP) or of PKA (6-Bnz-cAMP) for 4 days. Strips were mounted for isometric contraction experiments or analysed for the expression of contractile markers. Cell proliferation was measured and proliferative markers were analysed under similar conditions. KEY RESULTS: Activation of Epac and PKA prevented PDGF-induced ASM strip hypocontractility, and restored the expression of smooth muscle actin, myosin and calponin, which had been markedly diminished by PDGF. Epac and PKA activation inhibited the PDGF-induced ASM cell proliferation and G(1)/S phase transition and the expression and phosphorylation of cell cycle regulators. CONCLUSIONS AND IMPLICATIONS: Epac and PKA maintain a normally contractile ASM phenotype in a mitogenic environment, suggesting that specific activators of Epac and PKA may be beneficial in the treatment of airway remodelling in asthma.

cAMP inhibits modulation of airway smooth muscle phenotype via the exchange protein activated by cAMP (Epac) and protein kinase A.

BACKGROUND AND PURPOSE: Changes in airway smooth muscle (ASM) phenotype may contribute to the pathogenesis of airway disease. Platelet-derived growth factor (PDGF) switches ASM from a contractile to a proliferative, hypo-contractile phenotype, a process requiring activation of extracellular signal-regulated kinase (ERK) and p70(S6) Kinase (p70(S6K) ). The effects of cAMP-elevating agents on these processes is unknown. Here, we investigated the effects of cAMP elevation by prostaglandin E(2) (PGE(2) ) and the activation of the cAMP effectors, protein kinase A (PKA) and exchange protein activated by cAMP (Epac) on PDGF-induced phenotype switching in bovine tracheal smooth muscle (BTSM). EXPERIMENTAL APPROACH: Effects of long-term treatment with the PGE(2) analogue 16,16-dimethyl-PGE(2) , the selective Epac activator, 8-pCPT-2'-O-Me-cAMP and the selective PKA activator, 6-Bnz-cAMP were assessed on the induction of a hypo-contractile, proliferative BTSM phenotype and on activation of ERK and p70(S6K) , both induced by PDGF. KEY RESULTS: Treatment with 16,16-dimethyl-PGE(2) inhibited PDGF-induced proliferation of BTSM cells and maintained BTSM strip contractility and contractile protein expression in the presence of PDGF. Activation of both Epac and PKA similarly prevented PDGF-induced phenotype switching and PDGF-induced activation of ERK. Interestingly, only PKA activation resulted in inhibition of PDGF-induced phosphorylation of p70(S6K) . CONCLUSIONS AND IMPLICATIONS: Our data indicate for the first time that both Epac and PKA regulated switching of ASM phenotype via differential inhibition of ERK and p70(S6K) pathways. These findings suggest that cAMP elevation may be beneficial in the treatment of long-term changes in airway disease.

Epac as a novel effector of airway smooth muscle relaxation.

Dysfunctional regulation of airway smooth muscle tone is a feature of obstructive airway diseases such as asthma and chronic obstructive pulmonary disease. Airway smooth muscle contraction is directly associated with changes in the phosphorylation of myosin light chain (MLC), which is increased by Rho and decreased by Rac. Although cyclic adenosine monophosphate (cAMP)-elevating agents are believed to relieve bronchoconstriction mainly via activation of protein kinase A (PKA), here we addressed the role of the novel cAMP-mediated exchange protein Epac in the regulation of airway smooth muscle tone. Isometric tension measurements showed that specific activation of Epac led to relaxation of guinea pig tracheal preparations pre-contracted with methacholine, independently of PKA. In airway smooth muscle cells, Epac activation reduced methacholine-induced MLC phosphorylation. Moreover, when Epac was stimulated, we observed a decreased methacholine-induced RhoA activation, measured by both stress fibre formation and pull-down assay whereas the same Epac activation prevented methacholine-induced Rac1 inhibition measured by pull-down assay. Epac-driven inhibition of both methacholine-induced muscle contraction by Toxin B-1470, and MLC phosphorylation by the Rac1-inhibitor NSC23766, were significantly attenuated, confirming the importance of Rac1 in Epac-mediated relaxation. Importantly, human airway smooth muscle tissue also expresses Epac, and Epac activation both relaxed pre-contracted human tracheal preparations and decreased MLC phosphorylation. Collectively, we show that activation of Epac relaxes airway smooth muscle by decreasing MLC phosphorylation by skewing the balance of RhoA/Rac1 activation towards Rac1. Therefore, activation of Epac may have therapeutical potential in the treatment of obstructive airway diseases.

The role of Epac proteins, novel cAMP mediators, in the regulation of immune, lung and neuronal function.

Chronic degenerative inflammatory diseases, such as chronic obstructive pulmonary disease and Alzheimer's dementia, afflict millions of people around the world, causing death and debilitation. Despite the global impact of these diseases, there have been few innovative breakthroughs into their cause, treatment or cure. As with many debilitating disorders, chronic degenerative inflammatory diseases may be associated with defective or dysfunctional responses to second messengers, such as cyclic adenosinemonophosphate (cAMP). The identification of the cAMP-activated guanine nucleotide exchange factors for Ras-like GTPases, Epac1 (also known as cAMP-GEF-I) and Epac2 (also known as cAMP-GEF-II), profoundly altered the prevailing assumptions concerning cAMP signalling, which until then had been solely associated with protein kinase A (PKA). Studies of the molecular mechanisms of Epac-related signalling have demonstrated that these novel cAMP sensors regulate many physiological processes either alone and/or in concert with PKA. These include calcium handling, cardiac and smooth muscle contraction, learning and memory, cell proliferation and differentiation, apoptosis, and inflammation. The diverse signalling properties of cAMP might be explained by spatio-temporal compartmentalization, as well as A-kinase anchoring proteins, which seem to coordinate Epac signalling networks. Future research should focus on the Epac-regulated dynamics of cAMP, and, hopefully, the development of compounds that specifically interfere with the Epac signalling system in order to determine the precise significance of Epac proteins in chronic degenerative inflammatory disorders.

PKA and Epac cooperate to augment bradykinin-induced interleukin-8 release from human airway smooth muscle cells.

BACKGROUND: Airway smooth muscle contributes to the pathogenesis of pulmonary diseases by secreting inflammatory mediators such as interleukin-8 (IL-8). IL-8 production is in part regulated via activation of Gq-and Gs-coupled receptors. Here we study the role of the cyclic AMP (cAMP) effectors protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac1 and Epac2) in the bradykinin-induced IL-8 release from a human airway smooth muscle cell line and the underlying molecular mechanisms of this response. METHODS: IL-8 release was assessed via ELISA under basal condition and after stimulation with bradykinin alone or in combination with fenoterol, the Epac activators 8-pCPT-2'-O-Me-cAMP and Sp-8-pCPT-2'-O-Me-cAMPS, the PKA activator 6-Bnz-cAMP and the cGMP analog 8-pCPT-2'-O-Me-cGMP. Where indicated, cells were pre-incubated with the pharmacological inhibitors Clostridium difficile toxin B-1470 (GTPases), U0126 (extracellular signal-regulated kinases ERK1/2) and Rp-8-CPT-cAMPS (PKA). The specificity of the cyclic nucleotide analogs was confirmed by measuring phosphorylation of the PKA substrate vasodilator-stimulated phosphoprotein. GTP-loading of Rap1 and Rap2 was evaluated via pull-down technique. Expression of Rap1, Rap2, Epac1 and Epac2 was assessed via western blot. Downregulation of Epac protein expression was achieved by siRNA. Unpaired or paired two-tailed Student's t test was used. RESULTS: The beta2-agonist fenoterol augmented release of IL-8 by bradykinin. The PKA activator 6-Bnz-cAMP and the Epac activator 8-pCPT-2'-O-Me-cAMP significantly increased bradykinin-induced IL-8 release. The hydrolysis-resistant Epac activator Sp-8-pCPT-2'-O-Me-cAMPS mimicked the effects of 8-pCPT-2'-O-Me-cAMP, whereas the negative control 8-pCPT-2'-O-Me-cGMP did not. Fenoterol, forskolin and 6-Bnz-cAMP induced VASP phosphorylation, which was diminished by the PKA inhibitor Rp-8-CPT-cAMPS. 6-Bnz-cAMP and 8-pCPT-2'-O-Me-cAMP induced GTP-loading of Rap1, but not of Rap2. Treatment of the cells with toxin B-1470 and U0126 significantly reduced bradykinin-induced IL-8 release alone or in combination with the activators of PKA and Epac. Interestingly, inhibition of PKA by Rp-8-CPT-cAMPS and silencing of Epac1 and Epac2 expression by specific siRNAs largely decreased activation of Rap1 and the augmentation of bradykinin-induced IL-8 release by both PKA and Epac. CONCLUSION: Collectively, our data suggest that PKA, Epac1 and Epac2 act in concert to modulate inflammatory properties of airway smooth muscle via signaling to the Ras-like GTPase Rap1 and to ERK1/2.

Monomeric G-proteins as signal transducers in airway physiology and pathophysiology.

Monomeric G-proteins, also referred to as small GTPases, function as biological hubs being activated by extracellular stimuli and regulate downstream signalling events, which result in different cellular responses. The importance of these mechanisms is mirrored by the fact that several pathological conditions, including allergic asthma, are associated with derailed GTPases signalling. For this reason attention has been focused on the role of monomeric G-proteins and their effectors in airway (patho)physiology. In this article we review our current knowledge on the regulation and functions of Ras and Rho GTPase signalling under physiological and pathophysiological conditions in the pulmonary system. Based on recent findings concerning novel regulatory proteins for Ras family members, we further discuss potential future directions for therapeutical interventions in asthma.

Pharmacology of airway smooth muscle proliferation.

Airway smooth muscle thickening is a pathological feature that contributes significantly to airflow limitation and airway hyperresponsiveness in asthma. Ongoing research efforts aimed at identifying the mechanisms responsible for the increased airway smooth muscle mass have indicated that hyperplasia of airway smooth muscle, due in part to airway myocyte proliferation, is likely a major factor. Airway smooth muscle proliferation has been studied extensively in culture and in animal models of asthma, and these studies have revealed that a variety of receptors and mediators contributes to this response. This review aims to provide an overview of the receptors and mediators that control airway smooth muscle cell proliferation, with emphasis on the intracellular signalling mechanisms involved.

Epac: effectors and biological functions.

Epac1 (also known as cAMP-GEF-I) and Epac2 (also known as cAMP-GEF-II) are cyclic AMP-activated guanine nucleotide exchange factors for Ras-like GTPases. Since their discovery about 10 years ago, it is now accepted that Epac proteins are novel cAMP sensors that regulate several pivotal cellular processes, including calcium handling, cell proliferation, cell survival, cell differentiation, cell polarization, cell-cell adhesion events, gene transcription, secretion, ion transport, and neuronal signaling. Recent studies even indicated that Epac proteins might play a role in the regulation of inflammation and the development of cardiac hypertrophy. Meanwhile, a plethora of diverse effectors of Epac proteins have been assigned, such as Ras and Rho GTPases, phospholiase C-epsilon, phospholipase D, mitogen-activated protein kinases, protein kinase B/Akt, ion channels, secretory-granule associated proteins and regulators of the actin-microtubule network, the latter probably involved in the spatiotemporal dynamics of Epac-related signaling. This review highlights multi-faceted effectors and diverse biological functions driven by Epac proteins that might explain certain controversial signaling properties of cAMP.