Impact of TGF-ß inhibition during acute exercise on Achilles tendon extracellular matrix.

The purpose of this study was to evaluate the role of TGF-ß1 in regulating tendon extracellular matrix after acute exercise. Wistar rats exercised (n = 15) on a treadmill for four consecutive days (60 min/day) or maintained normal cage activity. After each exercise bout, the peritendinous space of each Achilles tendon was injected with a TGF-ß1 receptor inhibitor or sham. Independent of group, tendons injected with inhibitor exhibited ~50% lower Smad 3 (Ser423/425) (P < 0.05) and 2.5-fold greater ERK1/2 phosphorylation (P < 0.05) when compared with sham (P < 0.05). Injection of the inhibitor did not alter collagen content in either group (P > 0.05). In exercised rats, hydroxylyslpyridinoline content and collagen III expression were lower (P < 0.05) in tendons injected with inhibitor when compared with sham. In nonexercised rats, collagen I and lysyl oxidase (LOX) expression was lower (P < 0.05) in tendons injected with inhibitor when compared with sham. Decorin expression was not altered by inhibitor in either group (P > 0.05). On the basis of evaluation of hematoxylin and eosin (H&E) stained cross sections, cell numbers were not altered by inhibitor treatment in either group (P > 0.05). Evaluation of H&E-stained sections revealed no effect of inhibitor on collagen fibril morphology. In contrast, scores for regional variation in cellularity decreased in exercised rats (P < 0.05). No differences in fiber arrangement, structure, and nuclei form were noted in either group (P > 0.05). Our findings suggest that TGF-ß1 signaling is necessary for the regulation of tendon cross-link formation, as well as collagen and LOX gene transcription in an exercise-dependent manner.

Postnatal Hyperplasic Effects of ActRIIB Blockade in a Severely Dystrophic Muscle.

The efficacy of two ActRIIB ligand-trapping agents (RAP-031 and RAP-435) in treating muscular dystrophy was examined by determining their morphological effects on the severely dystrophic triangularis sterni (TS) muscle of the mdx mouse, a model for Duchenne muscular dystrophy. These agents trap all endogenous ligands to the ActRIIB receptor and thereby block myostatin signaling in a highly selective manner. Short-term (1 month) and long-term (3 months) in vivo treatment of 1-month-old mdx mice increased myonuclei and fiber cross section (FCS) density but did not alter individual fiber size. Vehicle-treated mdx mice exhibited age-dependent increases in myonuclei and FCS density, and age-dependent reductions in centronucleation that were each enhanced by treatment with RAP-435. Distributions of FCS area (FCSA) in the mdx TS were 90% identical to those from untreated age-matched nondystrophic mice and were unaltered by the substantial fiber hyperplasia observed with age and RAP-435 treatment. These results were inconsistent with injury-induced fiber regeneration which produces altered FCSA distributions characterized by a distinct class of smaller regenerated fibers. Nondystrophic mice exhibited a constant postnatal density of fiber cross sections and myonuclei, and RAP-435 treatment of nondystrophic mice increased TS mean FCSA but had no effects on myonuclei or FCS density. These results demonstrating a continual postnatal proliferation and fusion of satellite cells and a response to myostatin blockade characteristic of developing prenatal muscle suggest that the lack of dystrophin directly results in unrestrained postnatal satellite cell activation that is not necessarily dependent upon prior fiber degeneration. J. Cell. Physiol. 232: 1774-1793, 2017. © 2016 Wiley Periodicals, Inc.

Agents Which Inhibit NF-κB Signaling Block Spontaneous Contractile Activity and Negatively Influence Survival of Developing Myotubes.

Inhibiting the NF-κB signaling pathway provides morphological and functional benefits for the mdx mouse, a model for Duchenne muscular dystrophy characterized by chronic elevations in the nuclear expression of p65, the transactivating component of the NF-κB complex. The purpose of this study was to examine p65 expression in nondystrophic and mdx myotubes using confocal immunofluorescence, and determine whether inhibitors of the NF-κB pathway alter myotube development. Primary cultures of nondystrophic and mdx myotubes had identical levels of nuclear and cytosolic p65 expression and exhibited equivalent responses to TNF-α, thus excluding the hypothesis that the lack of dystrophin is sufficient to induce increases in NF-κB signaling. The NF-κB inhibitors pyrrolidine dithiocarbamate (PDTC) and sulfasalazine decreased spontaneous contractile activity and reduced myotube viability in a dose- and time-dependent manner. Similarly, a vivo-morpholino designed to block translation of murine p65 (m-p65tb-vivomorph1) rapidly abolished spontaneous contractile activity, reduced p65 expression measured by confocal immunofluorescence, and induced cell death in primary cultures of nondystrophic and mdx myotubes. Similar effects on p65 immunofluorescence and cell viability were observed following m-p65tb-vivomorph1 exposure to spontaneously inactive C2C12 myotubes, while exposure to a control scrambled vivo morpholino had no effect. These results indicate a direct role of the NF-κB pathway in myotube development and identify a potential therapeutic limitation to the use of NF-κB inhibitors in treating Duchenne and related muscular dystrophies. J. Cell. Physiol. 231: 788-797, 2016. © 2015 Wiley Periodicals, Inc.

Membrane cholesterol affects stimulus-activity coupling in type 1, but not type 2, CCK receptors: use of cell lines with elevated cholesterol.

The lipid microenvironment of membrane proteins can affect their structure, function, and regulation. We recently described differential effects of acute modification of membrane cholesterol on the function of type 1 and 2 cholecystokinin (CCK) receptors. We now explore the regulatory impact of chronic cholesterol modification on these receptors using novel receptor-bearing cell lines with elevated membrane cholesterol. Stable CCK1R and CCK2R expression was established in clonal lines of 25RA cells having gain-of-function in SCAP [sterol regulatory element binding protein (SREBP) cleavage-activating protein] and SRD15 cells having deficiencies in Insig-1 and Insig-2 enzymes affecting HMG CoA reductase and SREBP. Increased cholesterol in the plasma membrane of these cells was directly demonstrated, and receptor binding and signaling characteristics were shown to reflect predicted effects on receptor function. In both environments, both types of CCK receptors were internalized and recycled normally in response to agonist occupation. No differences in receptor distribution within the membrane were appreciated at the light microscopic level in these CHO-derived cell lines. Fluorescence anisotropy was studied for these receptors occupied by fluorescent agonist and antagonist, as well as when tagged with YFP. These studies demonstrated increased anisotropy of the agonist ligand occupying the active state of the CCK1R in a cholesterol-enriched environment, mimicking fluorescence of the uncoupled, inactive state of this receptor, while there was no effect of increasing cholesterol on fluorescence at the CCK2R. These cell lines should be quite useful for examining the functional characteristics of potential drugs that might be used in an abnormal lipid environment.

Differential sensitivity of types 1 and 2 cholecystokinin receptors to membrane cholesterol.

Recent studies indicate that membrane cholesterol can associate with G protein-coupled receptors (GPCRs) and affect their function. Previously, we reported that manipulation of membrane cholesterol affects ligand binding and signal transduction of the type 1 cholecystokinin receptor (CCK1R), a Class A GPCR. We now demonstrate that the closely related type 2 cholecystokinin receptor (CCK2R) does not share this cholesterol sensitivity. The sequences of both receptors reveal almost identical cholesterol interaction motifs in analogous locations in transmembrane segments two, three, four, and five. The disparity in cholesterol sensitivity between these receptors, despite their close structural relationship, provides a unique opportunity to define the possible structural basis of cholesterol sensitivity of CCK1R. To evaluate the relative contributions of different regions of CCK1R to cholesterol sensitivity, we performed ligand binding studies and biological activity assays of wild-type and CCK2R/CCK1R chimeric receptor-bearing Chinese hamster ovary cells after manipulation of membrane cholesterol. We also extended these studies to site-directed mutations within the cholesterol interaction motifs. The results contribute to a better understanding of the structural requirements for cholesterol sensitivity in CCK1R and provides insight into the function of other cholesterol-sensitive Class A GPCRs.

Regulation of N-formyl peptide receptor signaling and trafficking by individual carboxyl-terminal serine and threonine residues.

Adaptation, defined as the diminution of receptor signaling in the presence of continued or repeated stimulation, is critical to cellular function. G protein-coupled receptors (GPCRs) undergo multiple adaptive processes, including desensitization and internalization, through phosphorylation of cytoplasmic serine and threonine residues. However, the relative importance of individual and combined serine and threonine residues to these processes is not well understood. We examined this mechanism in the context of the N-formyl peptide receptor (FPR), a well-characterized member of the chemoattractant/chemokine family of GPCRs critical to neutrophil function. To evaluate the contributions of individual and combinatorial serine and threonine residues to internalization, desensitization, and arrestin2 binding, 30 mutant forms of the FPR, expressed in the human promyelocytic U937 cell line, were characterized. We found that residues Ser(328), Ser(332), and Ser(338) are individually critical, and indeed sufficient, for internalization, desensitization, and arrestin2 binding, but that the presence of neighboring threonine residues can inhibit these processes. Additionally, we observed no absolute correlation between arrestin binding and either internalization or desensitization, suggesting the existence of arrestin-independent mechanisms for these processes. Our results suggest C-terminal serine and threonine residues of the FPR represent a combinatorial code, capable of both positively and negatively regulating signaling and trafficking. This study is among the first detailed analyses of a complex regulatory site in a GPCR, and provides insight into GPCR regulatory mechanisms.

Arrestin variants display differential binding characteristics for the phosphorylated N-formyl peptide receptor carboxyl terminus.

The phosphorylation-dependent binding of arrestins to cytoplasmic domains of G protein-coupled receptors (GPCRs) is thought to be a crucial step in receptor desensitization. In some GPCR systems, arrestins have also been demonstrated to be involved in receptor internalization, resensitization, and the activation of signaling cascades. The objective of the current study was to examine binding interactions of members of the arrestin family with the formyl peptide receptor (FPR), a member of the GPCR family of receptors. Peptides representing the unphosphorylated and phosphorylated carboxyl terminus of the FPR were synthesized and bound to polystyrene beads via a biotin/streptavidin interaction. Using fluorescein-conjugated arrestins, binding interactions between arrestins and the bead-bound FPR carboxyl terminus were analyzed by flow cytometry. Arrestin-2 and arrestin-3 bound to the FPR carboxyl-terminal peptide in a phosphorylation-dependent manner, with K(d) values in the micromolar range. Binding of visual arrestin, which binds rhodopsin with high selectivity, was not observed. Arrestin-2-(1--382) and arrestin-3-(1--393), truncated mutant forms of arrestin that display phosphorylation-independent binding to intact receptors, were also observed to bind the bead-bound FPR terminus in a phosphorylation-dependent manner, but with much greater affinity than the full-length arrestins, yielding K(d) values in the 5--50 nm range. Two additional arrestin mutants, which are full-length but display phosphorylation-independent binding to intact GPCRs, were evaluated for their binding affinity to the FPR carboxyl terminus. Whereas the single point mutant, arrestin-2 R169E, displayed an affinity similar to that of the full-length arrestins, the triple point mutant, arrestin-2 I386A/V387A/F388A, displayed an affinity more similar to that of the truncated forms of arrestin. The results suggest that the carboxyl terminus of arrestin is a critical determinant in regulating the binding affinity of arrestin for the phosphorylated domains of GPCRs.