Association of pro/anti-inflammatory cytokine gene variants in renal transplant patients with allograft outcome and cyclosporine immunosuppressant levels

T-helper (Th) type 1/Th2 cytokines are key mediators in induction/effecter phases of all immune and inflammatory responses playing role in acute/chronic renal allograft rejection. Association studies lead to identification of patient risk profiles enabling individualization of level of immunosuppressions. We investigated the association of allograft rejection with interleukin-2 (IL-2), IL-4, IL-6, tumor necrosis factor-α (TNF-α) −308, transforming growth factor-β (TGF-β) (C-del, codon 10 and 25) gene variants in 184 renal transplant recipients and 180 controls. These cytokine genotypes were also evaluated with cyclosporine levels (C2) at one month in 135 stable recipients. High producing genotypes B1B1 of IL-4 and AA of TNF-α α308 showed significant association with rejection of allograft. The dose-adjusted C2 levels were significantly lower in patients with the high producing genotype T/T of IL-2 and heterozygous G/C of TGF-β codon 25 (P = 0.012 and 0.010, respectively). Haplotype frequencies were comparable in subjects for TGF-β codon-10 and 25. Combined inter-gene interaction showed high risk for rejection in recipients with high producing genotype B1B1 of IL-4 and AA of TNF-α and high TNF-α (AA) with low TGF-β (CC or Pro/Pro). In conclusion, association of IL-4 VNTR and TNF-α −308 suggested the involvement of these cytokines contributing to pathogenesis of allograft rejection. Recipients with TT genotype of IL-2 and GC of TGF-β codon 25 having low C2 levels may require higher cyclosporine dosage. Combined analysis of gene-gene interaction demonstrated synergistic effect of cytokines increasing risk for rejection. Thus, this information may help in pre-assessment of allograft outcome and to optimize cyclosporine therapy in post-transplant patients

Vitamin D receptor as a therapeutic target for benign prostatic hyperplasia

The bioactive form of vitamin D, 1α, 25-dihydroxyvitamin D3 (1α, 25(OH)2D3), is a secosteroid hormone that binds to the vitamin D receptor (VDR), a member of the nuclear receptor super-family expressed in many cell types, and modulates a variety of biological functions. 1α, 25(OH)2D3 is essential for bone and mineral homeostasis, but also regulates growth and differentiation of multiple cell types, and displays immunoregulatory and anti-inflammatory activities. The antiproliferative, prodifferentiative, antibacterial, immunomodulatory and anti-inflammatory properties of synthetic VDR agonists could be exploited to treat a variety of chronic inflammatory and autoimmune diseases, including benign prostatic hyperplasia (BPH). It has been hypothesized that VDR may influence both the risk of a variety of diseases and their occurrence and prognosis. However, earlier studies investigating the associations between specific VDR polymorphisms and various diseases often show controversial results. We performed a systematic review of the current literature on vitamin D and BPH using the PubMed and Web of Knowledge databases. The aim of this review is to summarize the current knowledge on the utility of the VDR gene regarding prostate growth as well as the pathogenesis and treatment of BPH, a complex syndrome characterized by a static component related to prostate overgrowth, a dynamic component responsible for urinary storage symptoms, and an inflammatory component. Despite the massive advances in recent decades, further research is needed to fully characterize the exact underlying mechanisms of VDR action on BPH and to comprehend how these cellular changes translate into clinical development in physical concert

Protein Kinase A Activation Enhances β-Catenin Transcriptional Activity through Nuclear Localization to PML Bodies

<div><p>The Protein Kinase A (PKA) and Wnt signaling cascades are fundamental pathways involved in cellular development and maintenance. In the osteoblast lineage, these pathways have been demonstrated functionally to be essential for the production of mineralized bone. Evidence for PKA-Wnt crosstalk has been reported both during tumorigenesis and during organogenesis, and the nature of the interaction is thought to rely on tissue and cell context. In this manuscript, we analyzed bone tumors arising from mice with activated PKA caused by mutation of the PKA regulatory subunit Prkar1a. In primary cells from these tumors, we observed relocalization of β-catenin to intranuclear punctuate structures, which were identified as PML bodies. Cellular redistribution of β-catenin could be recapitulated by pharmacologic activation of PKA. Using 3T3-E1 pre-osteoblasts as a model system, we found that PKA phosphorylation sites on β-catenin were required for nuclear re-localization. Further, β-catenin's transport to the nucleus was accompanied by an increase in canonical Wnt-dependent transcription, which also required the PKA sites. PKA-Wnt crosstalk in the cells was bi-directional, including enhanced interactions between β-catenin and the cAMP-responsive element binding protein (CREB) and transcriptional crosstalk between the Wnt and PKA signaling pathways. Increases in canonical Wnt/β-catenin signaling were associated with a decrease in the activity of the non-canonical Wnt/Ror2 pathway, which has been shown to antagonize canonical Wnt signaling. Taken together, this study provides a new understanding of the complex regulation of the subcellular distribution of β-catenin and its differential protein-protein interaction that can be modulated by PKA signaling.</p></div

Stimulation of PKA by FSK increases β-catenin phosphorylation and nuclear relocalization in MC3T3-E1 cells.

<p>A. Protein lysates were prepared from MC3T3-E1 cells treated with vehicle for Forskolin (FSK) for the times indicated and blotted with the antibodies shown. Note the increases in pS133-CREB, pS552- and pS675- β-catenin at both timepoints without changes in total protein levels. Actin is shown as a loading control. 20 µg of protein were loaded per lane. B. Immunofluorescence tracking of β-catenin after vehicle (DMSO) or FSK treatment in MC3T3-E1 cells. The bottom row shows a merged images from the panels above. Scale bar: 10 µm.</p

β-catenin forms punctate nuclear lesions in response to PKA activation in primary cultures.

<p>Primary osteoblasts from wild type (WT) bones or from bone tumors arising in <i>Prkar1a^+/−</i> mice were studied by immunofluorescence for β-catenin (green). For reference, cell nuclei were stained with DAPI. The left column shows β-catenin, only, whereas the right column shows merger of the β-catenin and DAPI stains. Top) WT osteoblasts. Middle) Tumor osteoblasts. Bottom) WT osteoblasts treated with forskolin (FSK). Note the punctate nuclear localization of β-catenin observed in Tumor cells or WT cells treated with FSK. Magnification: 400x.</p

Distribution of TCF and CREB binding sites in genes with altered transcription in <i>Prkar1a^+/− bone tumors</i>^*.

<p>*Distribution of promoters with neither or both sites is NS. Distribution of Tcf sites vs. up- and down-regulated genes has p = 0.037 by Fisher's exact test. Distribution of CREB and TCF sites vs. up- and down-regulated genes shows p<0.0001 by Fisher's exact test.</p><p>Distribution of TCF and CREB binding sites in genes with altered transcription in <i>Prkar1a^+/− bone tumors</i>^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109523#nt101" target="_blank">*</a>}.</p

PKA activation represses Wnt5a/Ror2 pathway.

<p>A. and B. mRNA expression of Wnt5a and Ror2 was determined using QPCR analysis in MC3T3-E1 cells treated with FSK (A) or with <i>Prkar1a</i> knockdown (B) (** P<0.01 versus DMSO or control shRNA treated cells). Error bars represent standard deviation. C. 20 ug of protein lysates from MC3T3-E1 cells were analyzed for Wnt5a/b by Western blotting. Actin was used as the internal control. This experiment was repeated at least twice with similar results, and a representative blot is shown.</p

PKA activation promotes nuclear relocalization of phospho-β-catenin.

<p>A. Immunofluorescence for pS675-β-catenin (green), PML (red), with DAPI nuclear staining (blue) in MC3T3-E1 cells treated with vehicle (DMSO) or FSK. Note the nuclear accumulation of phospho-β-catenin in response to FSK. B. MC3T3-E1 cells were treated with vehicle or FSK and nuclear and cytosolic protein fractions prepared and blotted for the proteins shown. Specificity of the fractionation is demonstrated by blotting for Lamin A (nuclear marker) and α-tubulin (cytosolic marker). Note the enhanced phospho-β-catenin only in the nuclear fraction in response to FSK. 8 µg of nuclear and 20 µg of cytosolic protein were loaded per lane. C. Control or Prkar1a-knockdown MC3T3-E1 cells were studied by IF as in panel A. Scale bar for all images: 10 µm.</p