IL-17-producing cells in ankylosing spondylitis patients show gender-based differences in gene expression.

OBJECTIVES: Gender has been shown to impact disease expression in ankylosing spondylitis (AS) and Th17 cells play a key role in AS pathogenesis. To better understand what Th17-associated immune pathways are different between men and women, we compared the transcriptome of IL-17-enriched peripheral blood mononuclear cells (PBMCs) in male and female AS patients, with a particular focus on inflammatory cytokine genes. METHODS: PBMCs were collected from 10 female and 11 male AS patients at the Clinical Research Unit of MetroHealth Medical Center. IL-17-enriched PBMCs were isolated and stimulated with CytoStim. RNA-sequencing (RNA-seq) was performed on the samples, and the data were analysed using iPathwayGuide. Inflammatory markers and genes related to Th17 differentiation and function were identified based on previous studies. RESULTS: RNA-seq identified 12,893 genes with 2,851 genes with p-values <0.05 with distinct patterns of gene expression between male and female AS patients. TGF-ß, PGE2, and S100 proteins were significantly upregulated in males. Levels of IL-12B, a Th17 inducer, were lower in males compared to females. Additionally, receptors of IL-6, 12, 23, TGF-ß, and PGE2 were downregulated in males, except for IL-17RC, which was upregulated. Genes involved in Th17 differentiation showed differential expression between genders, with elevated expression of BATF, SOCS1, NKD2, and ARID5A in men and decreased expression of FOXO1. CONCLUSIONS: Transcriptomic analysis revealed that male AS patients exhibit distinct expression patterns of IL-17 pro-inflammatory genes, which may contribute to the phenotypic differences observed between genders in AS.

Optimization of Human Mesenchymal Stem Cells for Rheumatoid Arthritis: Implications for Improved Therapeutic Outcomes.

OBJECTIVE: Seropositive rheumatoid arthritis (RA) is a chronic autoimmune disease that is rarely "cured." Human mesenchymal stem cells (hMSCs) are known to reduce inflammation and restore immune homeostasis. However, methods for predicting therapeutic hMSC potency have not been established. The goal of these studies was to use and refine an ex vivo functional assay that determines potency of hMSCs and can then be validated in clinical trials as a potency measure of hMSCs used therapeutically to treat RA. METHODS: Allogeneic hMSCs were cytokine-stimulated, and a conditioned medium (CM) was harvested. The CM was tested for the potential to attenuate RA CD4+ T cell proliferation using suppression assays. Indoleamine 2, 3-dioxygenase (IDO) mRNA, and protein were quantified in hMSCs as a measure to compare hMSCs across (prior) studies. RESULTS: To mimic a proinflammatory environment that resembles that in RA, interleukin-1(IL1ß), tumor necrosis factor α (TNFα), and interferon γ (IFNγ) (alone or in combination) were used to precondition hMSCs. Treating hMSCs with a combination of these cytokines generated a CM "secretome" that suppressed T cell proliferation between 70 and 83%. Forty-eight hours of cytokine preconditioning hMSCs was required to maximize this effect. T cell suppression positively correlated with increases in hMSC cellular IDO mRNA and protein. CONCLUSION: By standardizing assays to measure hMSC effects, their potency on T cell suppression can be quantified. These studies demonstrate that hMSCs can be compared functionally to identify optimal preparation(s) for therapeutic use in RA and that the potency of hMSC-dependent T cell suppression may differ between hMSC donors. Clinical studies are warranted to validate the hypothesis that ex vivo potency in suppressing T cells will positively correlate with a reduction in RA disease activity and increase in immunological quiescence.

Progerin sequestration of PCNA promotes replication fork collapse and mislocalization of XPA in laminopathy-related progeroid syndromes.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder that is caused by a point mutation in the LMNA gene, resulting in production of a truncated farnesylated-prelamin A protein (progerin). We previously reported that XPA mislocalized to the progerin-induced DNA double-strand break (DSB) sites, blocking DSB repair, which led to DSB accumulation, DNA damage responses, and early replication arrest in HGPS. In this study, the XPA mislocalization to DSBs occurred at stalled or collapsed replication forks, concurrent with a significant loss of PCNA at the forks, whereas PCNA efficiently bound to progerin. This PCNA sequestration likely exposed ds-ssDNA junctions at replication forks for XPA binding. Depletion of XPA or progerin each significantly restored PCNA at replication forks. Our results suggest that although PCNA is much more competitive than XPA in binding replication forks, PCNA sequestration by progerin may shift the equilibrium to favor XPA binding. Furthermore, we demonstrated that progerin-induced apoptosis could be rescued by XPA, suggesting that XPA-replication fork binding may prevent apoptosis in HGPS cells. Our results propose a mechanism for progerin-induced genome instability and accelerated replicative senescence in HGPS.-Hilton, B. A., Liu, J., Cartwright, B. M., Liu, Y., Breitman, M., Wang, Y., Jones, R., Tang, H., Rusinol, A., Musich, P. R., Zou, Y. Progerin sequestration of PCNA promotes replication fork collapse and mislocalization of XPA in laminopathy-related progeroid syndromes.

Development of a Functional Biomarker for Use in Cell-Based Therapy Studies in Seropositive Rheumatoid Arthritis.

UNLABELLED: Cell-based therapy has potential therapeutic value in autoimmune diseases such as rheumatoid arthritis (RA). In RA, reduction of disease activity has been associated with improvement in the function of regulatory T cells (Treg) and attenuated responses of proinflammatory effector T cells (Teff). Mesenchymal stem cells (MSCs) and related multipotent adult progenitor cells (MAPC) have strong anti-inflammatory and immunomodulatory properties and may be able to "reset" the immune system to a pre-RA state. MAPC are MSC-like cells that are slightly earlier in lineage, have greater expansion capacity, and can be used as "off-the-shelf" therapy. Assessment of cell-based therapy to treat arthritis and related diseases is limited by the lack of available biological correlates that can be measured early on and indicate treatment response. We set out to develop a functional measure that could be used ex vivo as a biomarker of response. We were able to demonstrate that MAPC products could inhibit Teff responses from patients with active RA and that Treg from RA patients suppressed Teff. This assay used ex vivo can be used with MAPC or Treg alone or in combination and reflects the overall level of Teff suppression. Use of a novel functional biomarker as an exploratory endpoint in trials of cell-based therapy should be of value to detect biological outcomes at a point prior to the time that clinical response might be observed. SIGNIFICANCE: Therapy with mesenchymal stem cells and related multipotent adult progenitor cells is immune modifying in a variety of diseases. There is interest in using cell-based therapy in rheumatoid arthritis (RA) to induce tolerance and "reset" the immune system to its pre-RA state. In a clinical trial, it should be known as soon as possible if there is a chance of response. A biomarker has been developed that permits measurement of the effects of cell-based therapy on effector T cell function.

Silent scaffolds: inhibition OF c-Jun N-terminal kinase 3 activity in cell by dominant-negative arrestin-3 mutant.

We established a new in vivo arrestin-3-JNK3 interaction assay based on bioluminescence resonance energy transfer (BRET) between JNK3-luciferase and Venus-arrestins. We tested the ability of WT arrestin-3 and its 3A mutant that readily binds ß2-adrenergic receptors as well as two mutants impaired in receptor binding, Δ7 and KNC, to directly bind JNK3 and to promote JNK3 phosphorylation in cells. Both receptor binding-deficient mutants interact with JNK3 significantly better than WT and 3A arrestin-3. WT arrestin-3 and Δ7 mutant robustly promoted JNK3 activation, whereas 3A and KNC mutants did not. Thus, receptor binding, JNK3 interaction, and JNK3 activation are three distinct arrestin functions. We found that the KNC mutant, which tightly binds ASK1, MKK4, and JNK3 without facilitating JNK3 phosphorylation, has a dominant-negative effect, competitively decreasing JNK activation by WT arrestin-3. Thus, KNC is a silent scaffold, a novel type of molecular tool for the suppression of MAPK signaling in living cells.

The effect of arrestin conformation on the recruitment of c-Raf1, MEK1, and ERK1/2 activation.

Arrestins are multifunctional signaling adaptors originally discovered as proteins that "arrest" G protein activation by G protein-coupled receptors (GPCRs). Recently GPCR complexes with arrestins have been proposed to activate G protein-independent signaling pathways. In particular, arrestin-dependent activation of extracellular signal-regulated kinase 1/2 (ERK1/2) has been demonstrated. Here we have performed in vitro binding assays with pure proteins to demonstrate for the first time that ERK2 directly binds free arrestin-2 and -3, as well as receptor-associated arrestins-1, -2, and -3. In addition, we showed that in COS-7 cells arrestin-2 and -3 association with ß(2)-adrenergic receptor (ß2AR) significantly enhanced ERK2 binding, but showed little effect on arrestin interactions with the upstream kinases c-Raf1 and MEK1. Arrestins exist in three conformational states: free, receptor-bound, and microtubule-associated. Using conformationally biased arrestin mutants we found that ERK2 preferentially binds two of these: the "constitutively inactive" arrestin-Δ7 mimicking microtubule-bound state and arrestin-3A, a mimic of the receptor-bound conformation. Both rescue arrestin-mediated ERK1/2/activation in arrestin-2/3 double knockout fibroblasts. We also found that arrestin-2-c-Raf1 interaction is enhanced by receptor binding, whereas arrestin-3-c-Raf1 interaction is not.

Identification of arrestin-3-specific residues necessary for JNK3 kinase activation.

Arrestins bind active phosphorylated G protein-coupled receptors, blocking G protein activation and channeling the signaling to G protein-independent pathways. Free arrestin-3 and receptor-bound arrestin-3 scaffold the ASK1-MKK4-JNK3 module, promoting JNK3 phosphorylation, whereas highly homologous arrestin-2 does not. Here, we used arrestin-2/3 chimeras and mutants to identify key residues of arrestin-3 responsible for its ability to facilitate JNK3 activation. Our data demonstrate that both arrestin domains are involved in JNK3 activation, with the C-terminal domain being more important than the N-terminal domain. We found that Val-343 is the key contributor to this function, whereas Leu-278, Ser-280, His-350, Asp-351, His-352, and Ile-353 play supporting roles. We also show that the arrestin-3-specific difference in the arrangement of the ß-strands in the C-terminal domain that underlies its lower selectivity for active phosphoreceptors does not play an appreciable role in its ability to enhance JNK3 activation. Importantly, the strength of the binding of ASK1 or JNK3, as revealed by the efficiency of co-immunoprecipitation, does not correlate with the ability of arrestin proteins to promote ASK1-dependent JNK3 phosphorylation. Thus, multiple residues on the non-receptor-binding side of arrestin-3 are crucial for JNK3 activation, and this function and the receptor-binding characteristics of arrestin can be manipulated independently by targeted mutagenesis.

A single mutation in arrestin-2 prevents ERK1/2 activation by reducing c-Raf1 binding.

Arrestins regulate the signaling and trafficking of G protein-coupled receptors (GPCRs). GPCR complexes with both nonvisual arrestins channel signaling to G protein-independent pathways, one of which is the activation of extracellular signal regulated kinase 1/2 (ERK1/2). Here we used alanine-scanning mutagenesis of residues on the nonreceptor-binding surface conserved between arrestin-2 and arrestin-3. We show that an Arg307Ala mutation significantly reduced arrestin-2 binding to c-Raf1, whereas the binding of the mutant to active phosphorylated receptor and downstream kinases MEK1 and ERK2 was not affected. In contrast to wild-type arrestin-2, the Arg307Ala mutant failed to rescue arrestin-dependent ERK1/2 activation via ß2-adrenergic receptor in arrestin-2/3 double knockout mouse embryonic fibroblasts. Thus, Arg307 plays a specific role in arrestin-2 binding to c-Raf1 and is indispensable in the productive scaffolding of c-Raf1-MEK1-ERK1/2 signaling cascade. Arg307Ala mutation specifically eliminates arrestin-2 signaling through ERK, which makes arrestin-2-Arg307Ala the first signaling-biased arrestin mutant constructed. In the crystal structure the side chain of homologous arrestin-3 residue Lys308 points in a different direction. Alanine substitution of Lys308 does not significantly affect c-Raf1 binding to arrestin-3 and its ability to promote ERK1/2 activation, suggesting that the two nonvisual arrestins perform the same function via distinct molecular mechanisms.

The armadillo repeat domain of the APC tumor suppressor protein interacts with Striatin family members.

Adenomatous polyposis coli (APC) is a multifunctional tumor suppressor protein that negatively regulates the Wnt signaling pathway. The APC gene is ubiquitously expressed in various tissues, especially throughout the large intestine and central nervous system. Mutations in the gene encoding APC have been found in most colorectal cancers and in other types of cancer. The APC gene product is a large multidomain protein that interacts with a variety of proteins, many of which bind to the well conserved armadillo repeat domain of APC. Through its binding partners, APC affects a large number of important cellular processes, including cell-cell adhesion, cell migration, organization of the actin and microtubule cytoskeletons, spindle formation and chromosome segregation. The molecular mechanisms that control these diverse APC functions are only partly understood. Here we describe the identification of an additional APC armadillo repeat binding partner - the Striatin protein. The Striatin family members are multidomain molecules that are mainly neuronal and are thought to function as scaffolds. We have found that Striatin is expressed in epithelial cells and co-localizes with APC in the epithelial tight junction compartment and in neurite tips of PC12 cells. The junctional localization of APC and Striatin is actin-dependent. Depletion of APC or Striatin affected the localization of the tight junction protein ZO-1 and altered the organization of F-actin. These results raise the possibility that the contribution of APC to cell-cell adhesion may be through interaction with Striatin in the tight junction compartment of epithelial cells.