Caveolins Muscle Their Way Into the Regulation of Cell Differentiation, Development and Function. Focus On “Muscle-Specific Interaction of Caveolin Isoforms (Cav-1, Cav-2 and Cav-3): Differential Complex Formation Between Caveolins in Fibroblastic Versus Muscle Cells

Since their initial characterization in the early 1990s, the functions ascribed to caveolin proteins have steadily increased in complexity and sophistication. Caveolins were initially identified as the main component of the “coat” of caveolae, vesicles that were originally described in the 1950s (22). Caveolae were initially considered to be vesicular structures that mediated transcytosis of macromolecules; caveolins were thus viewed as structural proteins that aided formation of the vesicle. By the mid-1990s, caveolae and their “siblings,” lipid rafts, rapidly became appreciated as “hot spots” for plasmalemmal signaling, with a newly recognized function as organizational or scaffolding proteins that attract and retain certain signaling moieties in efficient complexes. However, in recent years, evidence has steadily mounted to support the notion that caveolins are much more than simply structural components of vesicles or docking sites for signaling molecules. In fact, caveolins are now acknowledged to be critical regulators of several signaling pathways that control cell development, differentiation, and proliferation

Comparison of Functional Antagonism Between Isoproterenol and M2 Muscarinic Receptors in Guinea Pig Ileum and Trachea

The ability of the M2 muscarinic receptor to mediate an inhibition of the relaxant effects of forskolin and isoproterenol was investigated in guinea pig ileum and trachea. In some experiments, trachea was first treated with 4-diphenylacetoxy-Nmethylpiperidine (4-DAMP) mustard to inactivate M3 receptors. The contractile response to oxotremorine-M was measured subsequently in the presence of both histamine (10 mM) and isoproterenol (10 nM). Under these conditions, [[2-[(diethylamino) methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3b]- [1,4]benzodiazepine-6-one (AF-DX 116) antagonized the contractile response to oxotremorine-M in a manner consistent with an M3 mechanism. However, when the same experiment was repeated using forskolin (4 mM) instead of isoproterenol, the response to oxotremorine-M exhibited greater potency and was antagonized by AF-DX 116 in a manner consistent with an M2 mechanism. We also measured the effects of pertussis toxin treatment on the ability of isoproterenol to inhibit the contraction elicited by a single concentration of either histamine (0.3 mM) or oxotremorine-M (40 nM) in both the ileum and trachea. Pertussis toxin treatment had no significant effect on the potency of isoproterenol for inhibiting histamine-induced contractions in the ileum and trachea. In contrast, pertussis toxin treatment enhanced the relaxant potency of isoproterenol against oxotremorine-M-induced contractions in the ileum but not in the trachea. Also, pertussis toxin treatment enhanced the relaxant potency of forskolin against oxotremorine-M-induced contractions in the ileum and trachea. We investigated the relaxant potency of isoproterenol when very low, equi-effective (i.e., 20–34% of maximal response) concentrations of either histamine or oxotremorine-M were used to elicit contraction. Under these conditions, isoproterenol exhibited greater relaxant potency against histamine in the ileum but exhibited similar relaxant potencies against histamine and oxotremorine-M in the trachea. Following 4-DAMP mustard treatment, a low concentration of oxotremorine-M (10 nM) had no contractile effect in either the ileum or trachea. Nevertheless, in 4-DAMP mustard- treated tissue, oxotremorine-M (10 nM) reduced the relaxant potency of isoproterenol against histamine-induced contractions in the ileum, but not in the trachea. We conclude that in the trachea the M2 receptor mediates an inhibition of the relaxant effects of forskolin, but not isoproterenol, and the decreased relaxant potency of isoproterenol against contractions elicited by a muscarinic agonist relative to histamine is not due to activation of M2 receptors but rather to the greater contractile stimulus mediated by the M3 receptor compared with the H1 histamine receptor

Choreographing the Adenylyl Cyclase Signalosome: Sorting Out the Partners and the Steps

Adenylyl cyclases are a ubiquitous family of enzymes and are critical regulators of metabolic and cardiovascular function. Multiple isoforms of the enzyme are expressed in a range of tissues. However, for many processes, the adenylyl cyclase isoforms have been thought of as essentially interchangeable, with their impact more dependent on their common actions to increase intracellular cyclic adenosine monophosphate content regardless of the isoform involved. It has long been appreciated that each subfamily of isoforms demonstrate a specific pattern of “upstream” regulation, i.e., specific patterns of ion dependence (e.g., calcium-dependence) and specific patterns of regulation by kinases (protein kinase A (PKA), protein kinase C (PKC), raf). However, more recent studies have suggested that adenylyl cyclase isoform-selective patterns of signaling are a wide-spread phenomenon. The determinants of these selective signaling patterns relate to a number of factors, including: (1) selective coupling of specific adenylyl cyclase isoforms with specific G protein-coupled receptors, (2) localization of specific adenylyl cyclase isoforms in defined structural domains (AKAP complexes, caveolin/lipid rafts), and (3) selective coupling of adenylyl cyclase isoforms with specific downstream signaling cascades important in regulation of cell growth and contractility. The importance of isoform-specific regulation has now been demonstrated both in mouse models as well as in humans. Adenylyl cyclase has not been viewed as a useful target for therapeutic regulation, given the ubiquitous expression of the enzyme and the perceived high risk of off-target effects. Understanding which isoforms of adenylyl cyclase mediate distinct cellular effects would bring new significance to the development of isoform-specific ligands to regulate discrete cellular actions

Activation of Adenylyl Cyclase Reduces TGF-b Profibrotic Response in Osteoarthritic Fibroblast-like Synoviocytes

Purpose: The hallmarks of osteoarthritis (OA) include cartilage degeneration, bone remodeling and synovial fibrosis. Synovial fibrosis is characterized by excessive extracellular matrix (ECM) accumulation due to an imbalance in ECM production, in particular collagen, and its turnover. Transforming growth factor beta (TGF-β) and its associated signaling pathway mediated by ALK5, plays an important role in synovial fibrosis and blocking TGF-β’s effect prevents synovial fibrosis. Increasing intracellular cyclic AMP (cAMP) produces an antifibrotic effect in fibroblasts of multiple origins. Forskolin (FsK) is a naturally occurring diterpene in the roots of the Indian Coleus plant that activates adenylyl cyclase resulting in an elevation in intracellular cAMP levels. We hypothesized that FsK treatment results in an anti-fibrotic effect in TGF-β stimulated fibroblast-like synoviocytes (FLS) from patients with advanced OA. Methods: OA FLS (Cell Applications, USA) were harvested from patients undergoing total knee replacement. Cells were used between the 3rd and 6th passages for all experiments. OA FLS (300,000 cells per well) were treated with TGF-β (1ng/ml; R&D Systems) in the absence or presence of FsK (10μM; Sigma Aldrich) or SB431542, an ALK5 inhibitor (1μM, Sigma Aldrich) for 24 hours followed by RNA extraction using Trizol reagent and RNA concentrations were determined using a NanoDrop ND-2000 spectrophotometer. cDNA was synthesized using iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad, USA). Quantitative PCR (qPCR) was performed using TaqMan Fast Advanced Master Mix (Lifetechnologies, USA). The cycle threshold (Ct) value of genes of interest were normalized to the Ct value of GAPDH in the same sample, and the relative expression was calculated using the 2−ΔΔCt method. Genes of interest included collagens type 1 (COL1A1) and 3 (COL3A1), α2 smooth muscle actin (ACTA2), proteoglycan-4 (PRG4), matrix metalloproteinases 3, 9 and 13 (MMP3, MMP9 and MMP13), tissue inhibitor of metalloproteinase-1 (TIMP1) and aggrecanase-1 (ADAMTS4). Multiple group comparisons were performed by ANOVA or ANOVA on the ranks followed by pairwise group comparisons using Tukey\u27s test. Data is presented as the average ± S.D. of 3–6 independent experiments. Results:FsK treatment significantly reduced TGF-β induced expression of collagen type I (fig. 1A; p Conclusions: Using a model of TGF-β stimulated OA synovial fibroblasts, FsK treatment resulted in a reduction in the expression of collagen type I, a major component of fibrosis and α2 smooth muscle actin, a marker of fibroblast differentiation to myofibroblasts. To this end, FsK\u27s effect was comparable to the inhibition of intracellular TGF-β signaling. PRG4 regulates synovial proliferation and inflammation and FsK treatment enhanced PRG4 expression by OA fibroblasts. FsK reduced expression of matrix degrading enzymes, especially MMP3 and MMP9 involved in synovial proliferation, and MMP13 and ADAMTS4, involved in cartilage degradation. Increasing intracellular levels of cAMP in synovial fibroblasts may result in antifibrotic and chondroprotective effects in the joint

Palmitoylation at Cys 1145 in the Carboxyl Terminus of Human Type 6 Adenylyl Cyclase is Not Required for Targeting to Lipid Rafts and Caveolae

Palmitoylation is important for targeting certain membrane-associated and integral membrane proteins to lipid rafts and caveolae. Previous data have shown that adenylyl cyclase type 6 (AC6) is enriched in lipid rafts or caveolae while other isoforms of AC, such as AC2 and AC4, are excluded from these domains. We hypothesized that palmitoylation on a cysteine residue in the carboxyl terminus (C-terminus) of AC6 or other elements encoded in this region are required for AC6 expression in lipid rafts and caveolae. Thus, we expressed in Cos-7 cells epitope-tagged full length human AC6 and three different C-terminally truncated AC6 proteins, one (AC6 1-1148) retaining Cys 1145, a putative palmitoylation site, and two others (AC6 1-1144 and AC6 1-1127) lacking this residue. We used several approaches for assessing the subcellular localization of these expressed proteins: non-detergent biochemical isolation of lipid rafts and immunoblotting, immunoprecipitation of caveolin-1, Triton-X-100 insolubility, and immunoisolation of caveolae followed by adenylyl cyclase activity assays. We found that AC6 1-1144, AC6 1-1127 and AC6 1–1148 truncation proteins were each localized similarly to full-length AC6. We conclude that neither the putative palmitoylation site Cys 1145, nor other elements in the distal portion of the carboxyl terminus of AC6, are important for targeting of this effector enzyme to lipid rafts and caveolae

Editorial: Adenylyl Cyclase Isoforms as Potential Drug Targets

Editorial on the Research Topic Adenylyl cyclase isoforms as potential drug target

Phosphodiesterase isoforms and cAMP compartments in the development of new therapies for obstructive pulmonary diseases

The second messenger molecule 3′5′-cyclic adenosine monophosphate (cAMP) imparts several beneficial effects in lung diseases such as asthma, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). While cAMP is bronchodilatory in asthma and COPD, it also displays anti-fibrotic properties that limit fibrosis. Phosphodiesterases (PDEs) metabolize cAMP and thus regulate cAMP signaling. While some existing therapies inhibit PDEs, there are only broad family specific inhibitors. The understanding of cAMP signaling compartments, some centered around lipid rafts/caveolae, has led to interest in defining how specific PDE isoforms maintain these signaling microdomains. The possible altered expression of PDEs, and thus abnormal cAMP signaling, in obstructive lung diseases has been poorly explored. We propose that inhibition of specific PDE isoforms can improve therapy of obstructive lung diseases by amplifying specific cAMP signals in discreet microdomains

Inhibition of cPLA2-Mediated Arachidonic Acid Release by Cyclic AMP Defines a Negative Feedback Loop for P2Y-Receptor Activation in Mdck-D1 Cells

In Madin-Darby canine kidney D1cells extracellular nucleotides activate P2Y receptors that couple to several signal transduction pathways, including stimulation of multiple phospholipases and adenylyl cyclase. For one class of P2Y receptors, P2Y2 receptors, this stimulation of adenylyl cyclase and increase in cAMP occurs via the conversion of phospholipase A2 (PLA2)-generated arachidonic acid (AA) to prostaglandins (e.g. PGE2). These prostaglandins then stimulate adenylyl cyclase activity, presumably via activation of prostanoid receptors. In the current study we show that agents that increase cellular cAMP levels (including PGE2, forskolin, and the β-adrenergic agonist isoproterenol) can inhibit P2Y receptor-promoted AA release. The protein kinase A (PKA) inhibitor H89 blocks this effect, suggesting that this feedback inhibition occurs via activation of PKA. Studies with PGE2indicate that inhibition of AA release is attributable to inhibition of mitogen-activated protein kinase activity and in turn of P2Y receptor stimulated PLA2 activity. Although cAMP/PKA-mediated inhibition occurs for P2Yreceptor-promoted AA release, we did not find such inhibition for epinephrine (α1-adrenergic) or bradykinin-mediated AA release. Taken together, these results indicate that negative feedback regulation via cAMP/PKA-mediated inhibition of mitogen-activated protein kinase occurs for some, but not all, classes of receptors that promote PLA2 activation and AA release. We speculate that receptor-selective feedback inhibition occurs because PLA2activation by different receptors in Madin-Darby canine kidney D1 cells involves the utilization of different signaling components that are differentially sensitive to increases in cAMP or, alternatively, because of compartmentation of signaling components

Adenylyl Cyclase Isoform Specific Effects in Lipid Raft and Non-Lipid Raft Membrane Domains Regulate cAMP Compartmentation in Human Airway Smooth Muscle Cells

The formation of distinct macromolecular signaling complexes allows different G-protein coupled receptors to produce diverse functional responses, even while sharing a common second messenger such as cAMP. In human airway smooth muscle (HASM) cells, segregation of specific receptors into different membrane microdomains is thought to critically aid in generating compartmentalized cAMP responses. Whereas, E type prostaglandin receptors (EPRs) have been shown to be expressed in non-lipid raft domains of the plasma membrane, β-Adrenergic receptors (βARs) are predominantly expressed in caveolar lipid rafts. In the present study, we tested the hypothesis that adenylyl cyclase type 2 (AC2) preferentially couples to EPRs in a non-lipid raft domain, while adenylyl cyclase type 6 (AC6) selectively couples to βARs in lipid rafts. To do this, we examined the effect of overexpressing AC2 and AC6 on cAMP responses detected using genetically-encoded FRET-based biosensors targeted to lipid raft and non-lipid raft domains of the plasma membrane, as well as the bulk cytosolic compartment in HASM cells. This approach has the advantage of measuring the kinetics of cAMP production in living cells without the use of PDE inhibitors. Overexpression of AC2 enhanced the cAMP response to EPR activation associated with non-lipid raft domains, without significantly affecting responses detected elsewhere. AC2 overexpression also had no effect on cAMP responses to βAR activation detected in any subcellular location. These data confirm the hypothesis that AC2 couples exclusively with EPRs in a non-lipid raft membrane compartment. Overexpression of AC6, on the other hand, actually decreased the response to βAR stimulation associated with lipid rafts, without significantly affecting responses elsewhere. AC6 overexpression also had no effect on the responses to EPR activation detected anywhere in the cell. The ability of AC6 overexpression to inhibit βAR production of cAMP in lipid raft domains was reversed by inhibition of PDE4 activity with rolipram. It was also reversed by overexpression of Ht31 peptide, which disrupts the interaction of protein kinase A with A-kinase anchoring proteins (AKAPs). These results suggests that AC6 overexpression upregulates and/or recruits PKA and PDE4 activity, which then reduces βAR production of cAMP associated specifically with lipid raft domains