Systemic sclerosis biomarkers detection in the secretome of TGFß1-activated primary human lung fibroblasts.

TGFß1 is a profibrotic mediator that contributes to a broad spectrum of pathologies, including systemic sclerosis-associated pulmonary fibrosis (SSc-PF). However, the secretome of TGFß1-stimulated primary human normal lung (NL) fibroblasts has not been well characterized. Using fluorescent 2-dimensional gel electrophoresis (2D-PAGE) and differential gel electrophoresis (DIGE) followed by Mass Spectrometry, we identified 37 differentially secreted proteins in the conditioned media of TGFß1-activated NL fibroblasts and generated a protein-protein association network of the TGFß1 secretome using STRING. Functional enrichment revealed that several biological processes and pathways characteristic of PF were enriched. Additionally, by comparing the TGFß1 secretome of NL fibroblasts to proteomic biomarkers from biological fluids of systemic sclerosis (SSc) patients, we identified 11 overlapping proteins. Together our data validate the TGFß1-induced secretome of NL fibroblasts as a valid in vitro model that reflects SSc biomarkers and identify potential therapeutic targets for SSc-PF. SIGNIFICANCE: All proteins secreted by fibroblasts into the extracellular space, representing the secretome, promote cell-to-cell communication as well as tissue homeostasis, immune mechanisms, developmental regulation, proteolysis, development of the extracellular matrix (ECM) and cell adhesion. Therefore, it is crucial to understand how TGFß1, a well-known profibrotic cytokine, modulates the secretome of pulmonary fibroblasts, and how the TGFß1-induced secretome resembles biomarkers in SSc. Using functional enrichment analysis, key pathways and hub proteins can be identified and studied as potential therapeutic targets for pulmonary fibrosis.

Arrestin-dependent angiotensin AT1 receptor signaling regulates Akt and mTor-mediated protein synthesis.

Control of protein synthesis is critical to both cell growth and proliferation. The mammalian target of rapamycin (mTOR) integrates upstream growth, proliferation, and survival signals, including those transmitted via ERK1/2 and Akt, to regulate the rate of protein translation. The angiotensin AT1 receptor has been shown to activate both ERK1/2 and Akt in arrestin-based signalsomes. Here, we examine the role of arrestin-dependent regulation of ERK1/2 and Akt in the stimulation of mTOR-dependent protein translation by the AT1 receptor using HEK293 and primary vascular smooth muscle cell models. Nascent protein synthesis stimulated by both the canonical AT1 receptor agonist angiotensin II (AngII), and the arrestin pathway-selective agonist [Sar(1)-Ile(4)-Ile(8)]AngII (SII), is blocked by shRNA silencing of ßarrestin1/2 or pharmacological inhibition of Akt, ERK1/2, or mTORC1. In HEK293 cells, SII activates a discrete arrestin-bound pool of Akt and promotes Akt-dependent phosphorylation of mTOR and its downstream effector p70/p85 ribosomal S6 kinase (p70/85S6K). In parallel, SII-activated ERK1/2 helps promote mTOR and p70/85S6K phosphorylation, and is required for phosphorylation of the known ERK1/2 substrate p90 ribosomal S6 kinase (p90RSK). Thus, arrestins coordinate AT1 receptor regulation of ERK1/2 and Akt activity and stimulate protein translation via both Akt-mTOR-p70/85S6K and ERK1/2-p90RSK pathways. These results suggest that in vivo, arrestin pathway-selective AT1 receptor agonists may promote cell growth or hypertrophy through arrestin-mediated mechanisms despite their antagonism of G protein signaling.

Fibroblasts in fibrosis: novel roles and mediators.

Fibroblasts are the most common cell type of the connective tissues found throughout the body and the principal source of the extensive extracellular matrix (ECM) characteristic of these tissues. They are also the central mediators of the pathological fibrotic accumulation of ECM and the cellular proliferation and differentiation that occurs in response to prolonged tissue injury and chronic inflammation. The transformation of the fibroblast cell lineage involves classical developmental signaling programs and includes a surprisingly diverse range of precursor cell types-most notably, myofibroblasts that are the apex of the fibrotic phenotype. Myofibroblasts display exaggerated ECM production; constitutively secrete and are hypersensitive to chemical signals such as cytokines, chemokines, and growth factors; and are endowed with a contractile apparatus allowing them to manipulate the ECM fibers physically to close open wounds. In addition to ECM production, fibroblasts have multiple concomitant biological roles, such as in wound healing, inflammation, and angiogenesis, which are each interwoven with the process of fibrosis. We now recognize many common fibroblast-related features across various physiological and pathological protracted processes. Indeed, a new appreciation has emerged for the role of non-cancerous fibroblast interactions with tumors in cancer progression. Although the predominant current clinical treatments of fibrosis involve non-specific immunosuppressive and anti-proliferative drugs, a variety of potential therapies under investigation specifically target fibroblast biology.

Angiotensin II activates NF-κB through AT1A receptor recruitment of ß-arrestin in cultured rat vascular smooth muscle cells.

Activation of the angiotensin type 1A receptor (AT1AR) in rat aorta vascular smooth muscle cells (RASMC) results in increased synthesis of the proinflammatory enzyme cyclooxygenase-2 (COX-2). We previously showed that nuclear localization of internalized AT1AR results in activation of transcription of the gene for COX-2, i.e., prostaglandin-endoperoxide synthase-2. Others have suggested that ANG II stimulation of COX-2 protein synthesis is mediated by NF-κB. The purpose of the present study was to examine the interrelationship between AT1AR activation, ß-arrestin recruitment, and NF-κB activation in the ability of ANG II to increase COX-2 protein synthesis in RASMC. In the present study we utilized RASMC, inhibitors of the NF-κB pathway, ß-arrestin knockdown, radioligand binding, immunoblotting, and immunofluorescence to characterize the roles of AT1AR internalization, NF-κB activation, and ß-arrestin in ANG II-induced COX-2 synthesis. Ro-106-9920 or parthenolide, agents that inhibit the initial steps of NF-κB activation, blocked ANG II-induced p65 NF-κB nuclear localization, COX-2 protein expression, ß-arrestin recruitment, and AT1AR internalization without inhibiting ANG II-induced p42/44 ERK activation. Curcumin, an inhibitor of NF-κB-induced transcription, blocked ANG II-induced COX-2 protein expression without altering AT1AR internalization, ANG II-induced p65 NF-κB nuclear localization, or p42/44 ERK activation. Small interfering RNA-induced knockdown of ß-arrestin-1 and -2 inhibited ANG II-induced p65 NF-κB nuclear localization. In vascular smooth muscle cells, internalization of the activated AT1AR mediated by ß-arrestins activates the NF-κB pathway, producing nuclear localization of the transcription factor and initiation of COX-2 protein synthesis, thereby linking internalization of the receptor with the NF-κB pathway.

The beta-arrestin pathway-selective type 1A angiotensin receptor (AT1A) agonist [Sar1,Ile4,Ile8]angiotensin II regulates a robust G protein-independent signaling network.

The angiotensin II peptide analog [Sar(1),Ile(4),Ile(8)]AngII (SII) is a biased AT(1A) receptor agonist that stimulates receptor phosphorylation, ß-arrestin recruitment, receptor internalization, and ß-arrestin-dependent ERK1/2 activation without activating heterotrimeric G-proteins. To determine the scope of G-protein-independent AT(1A) receptor signaling, we performed a gel-based phosphoproteomic analysis of AngII and SII-induced signaling in HEK cells stably expressing AT(1A) receptors. A total of 34 differentially phosphorylated proteins were detected, of which 16 were unique to SII and eight to AngII stimulation. MALDI-TOF/TOF mass fingerprinting was employed to identify 24 SII-sensitive phosphoprotein spots, of which three (two peptide inhibitors of protein phosphatase 2A (I1PP2A and I2PP2A) and prostaglandin E synthase 3 (PGES3)) were selected for validation and further study. We found that phosphorylation of I2PP2A was associated with rapid and transient inhibition of a ß-arrestin 2-associated pool of protein phosphatase 2A, leading to activation of Akt and increased phosphorylation of glycogen synthase kinase 3ß in an arrestin signalsome complex. SII-stimulated PGES3 phosphorylation coincided with an increase in ß-arrestin 1-associated PGES3 and an arrestin-dependent increase in cyclooxygenase 1-dependent prostaglandin E(2) synthesis. These findings suggest that AT(1A) receptors regulate a robust G protein-independent signaling network that affects protein phosphorylation and autocrine/paracrine prostaglandin production and that these pathways can be selectively modulated by biased ligands that antagonize G protein activation.

Isoform-specific uncoupling of the D2 dopamine receptors subtypes.

Dopaminergic transmission is fundamental to many neural pathways of clinical interest. We have analyzed the alternatively-spliced isoforms of the D(2) dopamine receptor, D(2) long (D(2l)) and D(2) short (D(2s)), which differ only by a 29-amino acid insertion in the third cytoplasmic loop. Well-known determinants for GPCR signal transduction--the third intracellular loop regions--were co-expressed with the wild-type receptors to test for their ability to antagonize parent receptor function. We found that the D(2l)-mediated inhibition of forskolin-stimulated adenylyl cyclase was blocked by the co-expression of the third cytoplasmic loop of D(2l). However, expression of the third cytoplasmic loop of D(2s) did not inhibit D(2l)-mediated signal transduction. Conversely, expression of the D(2s) third cytoplasmic loop antagonized the D(2s) receptor's function and the D(2l) third cytoplasmic loop did not. In contrast, expression of the alternatively-spliced insert region had no effect when co-expressed with either wild-type receptor isoform. These results suggest that the third cytoplasmic loops of each receptor adopt unique conformations and that the primary sequence of the insert region is not the basis for differences in signaling between D(2s) and D(2l). These findings further support previous studies suggesting that the D2 receptor isoforms use distinct signal transduction mechanisms.

Diversity in arrestin function.

The termination of heptahelical receptor signaling is a multilevel process coordinated, in large part, by members of the arrestin family of proteins. Arrestin binding to agonist-occupied receptors promotes desensitization by interrupting receptor-G protein coupling, while simultaneously recruiting machinery for receptor endocytosis, vesicular trafficking, and receptor fate determination. By simultaneously binding other proteins, arrestins also act as ligand-regulated scaffolds that recruit protein and lipid kinase, phosphatase, phosphodiesterase, and ubiquitin ligase activity into receptor-based multiprotein 'signalsome' complexes. Arrestin-binding thus 'switches' receptors from a transient G protein-coupled state to a persistent arrestin-coupled state that continues to signal as the receptor transits intracellular compartments. While it is clear that signalsome assembly has profound effects on the duration and spatial characteristics of heptahelical receptor signals, the physiologic functions of this novel signaling mechanism are poorly understood. Growing evidence suggests that signalsomes regulate such diverse processes as endocytosis and exocytosis, cell migration, survival, and contractility.

Angiotensin II-induced cyclooxygenase 2 expression in rat aorta vascular smooth muscle cells does not require heterotrimeric G protein activation.

Angiotensin II (AngII) initiates cellular effects via its G protein-coupled angiotensin 1 (AT(1)) receptor (AT(1)R). Previously, we showed that AngII-induced expression of the prostanoid-producing enzyme cyclooxygenase 2 (COX-2) was dependent upon nuclear trafficking of activated AT(1)R. In the present study, mastoparan (an activator of G proteins), suramin (an inhibitor of G proteins), 1-[6-[[17beta-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122; a specific inhibitor of phospholipase C), and sarcosine(1)-Ile(4)-Ile(8)-AngII (SII-AngII; a G protein-independent AT(1)R agonist) were used to determine the involvement of G proteins and AT(1A)R trafficking in AngII-stimulated COX-2 protein expression in human embryonic kidney-293 cells stably expressing AT(1A)/green fluorescent protein receptors and cultured vascular smooth muscle cells, respectively. Mastoparan alone stimulated release of intracellular calcium and increased COX-2 expression. Preincubation with mastoparan inhibited AngII-induced calcium signaling without altering AngII-induced AT(1A)R trafficking, p42/44 extracellular signal-regulated kinase (ERK) activation, or COX-2 expression. Suramin or U73122 had no significant effect on their own; they did not inhibit AngII-induced AT(1A)R trafficking, p42/44 ERK activation, or COX-2 expression; but they did inhibit AngII-induced calcium responses. SII-AngII stimulated AT(1A)R trafficking and increased COX-2 protein expression without activating intracellular calcium release. These data suggest that G protein activation results in increased COX-2 protein expression, but AngII-induced COX-2 expression seems to occur independently of G protein activation.

Investigation of the alternatively spliced insert region of the D2L dopamine receptor by epitope substitution.

Alternatively spliced variants of the D2 dopamine receptor have distinct neuronal function and localization. The long isoform (D2L) of this heptahelical transmembrane receptor differs from the short form only by the presence of a 29-amino acid insert in the third intracellular loop-a region known to be important for G protein coupling. Short and long isoforms have been shown to have distinct Galphai/o protein coupling specificities. However, the exact role of the alternatively spliced insert region in D2 dopamine receptor function needs a more comprehensive examination. One way to address this is to substitute the entire insert region with an equivalent length, yet nonhomologous protein sequence. This report demonstrates the feasibility of replacing the 29-amino acid insert with a hemagglutinin double epitope tag with no recognizable functional consequences. The D2L mutant is indistinguishable from the wild type D2L receptor in terms of its ligand binding characteristics, as well as two effector responses: the agonist-mediated inhibition of forskolin-stimulated cAMP production, and agonist-stimulated MAPK phosphorylation. These data demonstrate that the epitope substitution generates a functional receptor, and that the alternatively spliced insert region, itself, does not appear to play a direct role in signal transduction. The epitope substitution permits dissection of sequence-mediated effects from structural effects due to the presence of the alternatively spliced insert region. Thus, this new construct could be a valuable tool for the study of D2 receptor function.