STIMULATION OF PHOSPHOLIPASE C-BETA(2) BY RECOMBINANT GUANINE-NUCLEOTIDE-BINDING PROTEIN BETA-GAMMA DIMERS PRODUCED IN A BACULOVIRUS/INSECT CELL EXPRESSION SYSTEM - REQUIREMENT OF GAMMA-SUBUNIT ISOPRENYLATION FOR STIMULATION OF PHOSPHOLIPASE-C

Recombinant wild-type beta(1) gamma(1) dimers of signal-transducing guanine nucleotide-binding proteins (G proteins) and beta(1) gamma 1 dimers carrying a mutation known to block gamma-subunit isoprenylation (beta(1) gamma(1)C71S) were expressed in baculovirus-infected insect cells. Both wild-type and mutant beta(1) gamma(1) dimers were found in soluble fractions of infected cells upon subcellular fractionation. Anion exchange chromatographic and metabolic-radiolabeling studies revealed that the soluble beta(1) gamma(1) preparation contained approximately equal amounts of non-isoprenylated and isoprenylated beta(1) gamma(1) dimers. Soluble wild-type and mutant beta(1) gamma(1) dimers and native beta(1) gamma(1) dimers purified from bovine retina were reconstituted with recombinant phospholipase C-beta(2). Only isoprenylated beta(1) gamma(1) dimers were capable of stimulating phospholipase C-beta(2). The results show that gamma-subunit isoprenylation and/or additional post-translational processing of the protein are required for beta gamma subunit stimulation of phospholipase C.

Prokineticin ligands and receptors are expressed in the human fetal ovary and regulate germ cell expression of COX2

CONTEXT: Fetal ovarian development and primordial follicle formation underpin future female fertility. Prokineticin (PROK) ligands regulate cell survival, proliferation and angiogenesis in adult reproductive tissues including the ovary. However, their expression and function during fetal ovarian development remains unclear.

OBJECTIVE: To investigate expression and localization of the PROK ligands, receptors and their downstream transcriptional targets in the human fetal ovary.

SETTING: This study was conducted at the University of Edinburgh.

PARTICIPANTS: Ovaries were collected from 37 morphologically normal human fetuses.

DESIGN AND MAIN OUTCOME MEASURES: mRNA and protein expression of PROK ligands and receptors was determined in human fetal ovaries using qRT-PCR, immunoblotting and immunohistochemistry. Functional studies were performed using a human germ tumour cell line (TCam-2) stably transfected with PROKR1.

RESULTS: Expression of PROK1 and PROKR1 was significantly higher in mid-gestation ovaries (17-20 weeks) than at earlier gestations (8-11 and 14-16 weeks). PROK2 significantly increased across the gestations examined. PROKR2 expression remained unchanged. PROK ligand and receptor proteins were predominantly localised to germ cells (including oocytes within primordial follicles) and endothelial cells, indicating these cell types to be the targets of PROK signalling in the human fetal ovary. PROK1 treatment of a germ cell line stably-expressing PROKR1 resulted in ERK phosphorylation, and elevated COX2 expression.

CONCLUSIONS: Developmental changes in expression and regulation of COX2 and pERK by PROK1 suggest that PROK ligands may be novel regulators of germ cell development in the human fetal ovary, interacting within a network of growth and survival factors prior to primordial follicle formation.

Preproendothelin-1 expression in human mesangial cells: evidence for a p38 mitogen-activated protein kinase/protein kinases- C-dependent mechanism

Endothelin-1 (ET-1) has been implicated in the pathogenesis of renal inflammation. This study investigated the mechanisms underlying the synergistic upregulation of preproET-1 gene expression in human mesangial cells after co-stimulation with thrombin and tumor necrosis factor alpha (TNFalpha). Whereas thrombin induced a moderate upregulation of preproET-1 mRNA, co-stimulation with TNFalpha resulted in a strong and protracted upregulation of this mRNA species. Thrombin+TNFalpha-induced upregulation of preproET-1 expression was found to require p38 mitogen-activated protein kinase and protein kinases C, whereas activation of extracellular signal-regulated kinase, c-Jun-N-terminal kinase, or intracellular Ca(2+) release were not required. Actinomycin D chase experiments suggested that enhanced stability of preproET-1 mRNA did not account for the increase in transcript levels. PreproET-1 promoter analysis demonstrated that the 5'-flanking region of preproET-1 encompassed positive regulatory elements engaged by thrombin. Negative modulation of thrombin-induced activation exerted by the distal 5' portion of preproET-1 promoter (-4.4 kbp to 204 bp) was overcome by co-stimulation with TNFalpha, providing a possible mechanism underlying the synergistic upregulation of preproET-1 expression by these two agonists. In conclusion, human mesangial cell expression of preproET-1 may be increased potently in the presence of two common proinflammatory mediators, thereby providing a potential mechanism for ET-1 production in inflammatory renal disease.

Novel Protective Properties of IGFBP-3 Result in Enhanced Pericyte Ensheathment, Reduced Microglial Activation, Increased Microglial Apoptosis, and Neuronal Protection after Ischemic Retinal Injury

This study was conducted to determine the perivascular cell responses to increased endothelial cell expression of insulin-like growth factor binding protein-3 (IGFBP-3) in mouse retina. The contribution of bone marrow cells in the IGFBP-3-mediated response was examined using green fluorescent protein-positive (GFP(+)) adult chimeric mice subjected to laser-induced retinal vessel occlusion injury. Intravitreal injection of an endothelial-specific IGFBP-3-expressing plasmid resulted in increased differentiation of GF(P)+ hematopoietic stem cells (HSCs) into pericytes and astrocytes as determined by immunohistochemical analysis. Administration of IGFBP-3 plasmid to mouse pups that underwent the oxygen-induced retinopathy model resulted in increased pericyte ensheathment and reduced pericyte apoptosis in the developing retina. Increased IGFBP-3 expression reduced the number of activated microglial cells and decreased apoptosis of neuronal cells in the oxygen-induced retinopathy model. In summary, IGFBP-3 increased differentiation of GFP(+) HSCs into pericytes and astrocytes while increasing vascular ensheathment of pericytes and decreasing apoptosis of pericytes and retinal neurons. All of these cytoprotective effects exhibited by IGFBP-3 overexpression can result in a more stable retinal vascular bed. Thus, endothelial expression of IGFBP-3 may represent a physiologic response to injury and may represent a therapeutic strategy for the treatment of ischemic vascular eye diseases, such as diabetic retinopathy and retinopathy of prematurity. (Am J Pathol 2011, 178:1517-1524; DOI: 10.1016/j.ajpath.2010.12.031)

Nonapical and Cytoplasmic Expression of Interleukin-8, CXCR1, and CXCR2 Correlates with Cell Proliferation and Microvessel Density in Prostate Cancer

Purpose: We characterized interleukin-8 (IL-8) and IL-8 receptor expression (CXCR1 and CXCR2) in prostate cancer to address their significance to this disease. Experimental Design: Immunohistochemistry was conducted on 40 cases of human prostate biopsy containing histologically normal and neoplastic tissue, excised from patients with locally confined or invasive androgen-dependent prostate cancer, and 10 cases of transurethral resection of the prostate material from patients with androgen-independent disease. Results: Weak to moderate IL-8 expression was strictly localized to the apical membrane of normal prostate epithelium. In contrast, membranous expression of IL-8, CXCR1, and CXCR2 was nonapical in cancer cells of Gleason pattern 3 and 4, whereas circumferential expression was present in Gleason pattern 5 and androgen-independent prostate cancer. Each of IL-8, CXCR1, and CXCR2 were also increasingly localized to the cytoplasm of cancer cells in correlation with advancing stage of disease. Cytoplasmic expression (but not apical membrane expression) of IL-8 in Gleason pattern 3 and 4 cancer correlated with Ki-67 expression (R = 0.79; P <0.001), cyclin D1 expression (R = 0.79; P <0.001), and microvessel density (R = 0.81; P <0.001). In vitro studies on androgen-independent PC3 cells confirmed the mitogenic activity of IL-8, increasing the rate of cell proliferation through activation of both CXCR1 and CXCR2 receptors. Conclusions: We propose that the concurrent increase in IL-8 and IL-8 receptor expression in human prostate cancer induces autocrine signaling that may be functionally significant in initiating and promoting the progression of prostate cancer by underpinning cell proliferation and angiogenesis.