Regulation of scleral cell contraction by transforming growth factor-β and stress competing roles in myopic growth

Reduced extracellular matrix accumulation in the sclera of myopic eyes leads to increased ocular extensibility and is related to reduced levels of scleral transforming growth factor-β (TGF-β). The current study investigated the impact of this extracellular environment on scleral cell phenotype and cellular biomechanical characteristics. Scleral cell phenotype was investigated in vivo in a mammalian model of myopia using the myofibroblast marker, α-smooth muscle actin (α-SMA). In eyes developing myopia α-SMA levels were increased, suggesting increased numbers of contractile myofibroblasts, and decreased in eyes recovering from myopia. To understand the factors regulating this change in scleral phenotype, the competing roles of TGF-β and mechanical stress were investigated in scleral cells cultured in three-dimensional collagen gels. All three mammalian isoforms of TGF-β altered scleral cell phenotype to produce highly contractile, α-SMA-expressing myofibroblasts (TGF-β3 > TGF-β2 > TGF-β1). Exposure of cells to the reduced levels of TGF-β found in the sclera in myopia produced decreased cell-mediated contraction and reduced α-SMA expression. These findings are contrary to the in vivo gene expression data. However, when cells were exposed to both the increased stress and the reduced levels of TGF-β found in myopia, increased α-SMA expression was observed, replicating in vivo findings. These results show that although reduced scleral TGF-β is a major contributor to the extracellular matrix remodeling in the myopic eye, it is the resulting increase in scleral stress that dominates the competing TGF-β effect, inducing increased α-SMA expression and, hence, producing a larger population of contractile cells in the myopic eye.

Identification of novel genes differentially expressed in haemopoietic progenitor cells

The biochemical and molecular processes that maintain the stem cell pool, and govern the proliferation and differentiation of haemopoietic stem/progenitor cells (HSPCs) have been widely investigated but are incompletely understood. The purpose of this study was to identify and characterise novel genes that may play a part in regulating the mechanisms that control the proliferation, differentiation and self-renewal of human HSPCs. Reverse transcription differential display polymerase chain reaction (dd-PCR) was used to identify differences in gene expression between a HSPC population defined by expression of the CD34 phenotype, and the more mature CD34 depleted populations. A total of 6 differentially expressed complementary deoxyribonucleic acid (cDNA) sequences were identified. Four of these transcripts were homologous to well characterised genes, while two (band 1 and band 20) were homologous to unknown and uncharacterised partial gene sequences on the GenBank database and were thus chosen for further investigation. The partial cDNA sequences for band 1 and band 20 were designated ORP-3 and MERP-1 (respectively) due to homologies with other well-characterised gene families. Differential expression of the ORP-3 and MERP-1 genes was confirmed using Taqman™ real-time polymerase chain reaction (PCR) with 3 - 4-fold and 4-10 -fold higher levels in the CD34+ fractions of haemopoietic cells compared to CD34- populations respectively. Additionally, expression of both these genes was down regulated with proliferation and differentiation of CD34+ cells further confirming higher expression in a less differentiated subset of haemopoietic cells. The full coding sequences of ORP-3 and MERP-1 were elucidated using bioinformatics, rapid amplification of cDNA ends (RACE) and PCR amplification. The MERP-1 cDNA is 2600 nucleotides (nt) long, and localizes by bioinformatics to chromosome 7.. It consists of three exons and 2 introns spanning an entire length of 31.4 kilobases (kb). The MERP-1 open reading frame (ORF) codes for a putative 344 amino acid (aa) type II transmembrane protein with an extracellular C-terminal ependymin like-domain and an intracellular N-terminal sequence with significant homology to the cytoplasmic domains of members of the protocadherin family of transmembrane glycoproteins. Ependymins and protocadherins are well-characterised calcium-dependant cell adhesion glycoproteins. Although the function of MERP-1 remains to be elucidated, it is possible that MERP-1 like its homologues plays a role in calcium dependent cell adhesion. Differential expression of the MERP-1 gene in haemopoietic cells suggests a role in haemopoietic stem cell proliferation and differentiation, however, its broad tissue distribution implies that it may also play a role in many cell types. Characterization of the MERP-1 protein is required to elucidate these possible roles. The ORP-3 cDNA is 6631nt long, and localizes by bioinformatics to chromosome 7pl5-p21. It consists of 23 exons and 22 introns spanning an entire length of 183.5kb. The ORP-3 ORF codes for a putative 887aa protein which displays the consensus sequence for a highly conserved oxysterol-binding domain. Other well-characterised proteins expressing these domains have been demonstrated to bind oxysterols (OS) in a dose dependant fashion. OS are hydroxylated derivatives of cholesterol Their biological activities include inhibition of cholesterol biosynthesis and cell proliferation in a variety of cell types, including haemopoietic cells. Differential expression of the ORP-3 gene in haemopoietic cells suggests a possible role in the transduction of OS effects on haemopoietic cells, however, its broad tissue distribution implies that it may also play a role in many cell types. Further investigation of ORP-3 gene expression demonstrates a significant correlation with CD34+ sample purity, and 2-fold higher expression in a population of haemopoietic cells defined by the CD34+38- phenotype compared to more mature CD34+38+ cells. This finding, taken together with the previous observation of down-regulation of ORP-3 expression with proliferation and differentiation of CD34+ cells, indicates that ORP-3 expression may be higher in a less differentiated subset of cells with a higher proliferative capacity. This hypothesis is supported by the observation that expression of the ORP-3 gene is approximately 2-fold lower in differentiated HL60 promyelocytic cells compared to control, undifferentiated cells. ORP-3 expression in HL60 cells during normal culture conditions was also found to vary with expression positively correlated with cell number. This indicates a possible cell cycle effect on ORP-3 gene expression with levels highest when cell density, and therefore the percentage of cells in G(0)/G(1) phase of the cell cycle is highest. This observation also correlates with the observation of higher ORP-3 expression in CD34+38-cells, and in CD34+ and HL60 cells undergoing OS induced and camptothecin induced apoptosis that is preceded by cell cycle arrest at G(0)/G(1). Expression of the ORP-3 gene in CD34+ HSPCs from UCB was significantly decreased to approximately half the levels observed in control cells after 24 hours incubation in transforming growth factor beta-1 (TGFâl). As ≥90% of these cells are stimulated into cell cycle entry by TGFâl, this observation further supports the hypothesis that ORP-3 expression is highest when cells reside in the G(0)/G(1) phase of the cell cycle. Data obtained from investigation of ORP-3 gene expression in synchronised HL60 cells however does not support nor disprove this hypothesis. Culture of CD34+ enriched HSPCs and HL60 cells with 25-OHC significantly increased ORP-3 gene expression to approximately 1.5 times control levels. However, as 25-OHC treatment also increased the percentage of apoptotic cells in these experiments, it is not valid to make any conclusions regarding the regulation of ORP-3 gene expression by OS. Indeed, the observation that camptothecin induced apoptosis also increased ORP-3 gene expression in HL60 cells raises the possibility that up-regulation of ORP-3 gene expression is also associated with apoptosis, Taken together, expression of the ORP-3 gene appears to be regulated by differentiation and apoptosis of haemopoietic progenitors, and may also be positively associated with proliferative and G(0)/G(1) cell cycle status indicating a possible role in all of these processes. Given the important regulatory role of apoptosis in haemopoiesis and differential expression of the ORP-3 gene in haemopoietic progenitors, final investigations were conducted to examine the effects OS on human HSPCs. Granulocyte/macrophage colony forming units (CFU-GM) generated from human bone marrow (ABM) and umbilical cord blood (UCB) were grown in the presence of varying concentrations of three different OS - 7keto-cholesterol (7K-C), 7beta-hydroxycholesterol (7p-OHC) and 25-hydroxycholesterol (25-OHC). Similarly, the effect of OS on HL60 and CD34+ cells was investigated using annexin-V staining and flow cytometry to measure apoptosis. Reduction of nitroblue tetrazolium (NBT) was used to assess differentiative status of HL60 cells. CFU-GM from ABM and HL60 growth was inhibited by all three OS tested, with 25-OHC being the most potent. 25-OHC inhibited ≥50% of bone marrow CFU-GM and ≥95% of HL60 cell growth at a level of 1 ug/ml. Compared to UCB, CFU-GM derived from ABM were more sensitive to the effects of all OS tested. Only 25-OHC and 7(5-OHC significantly inhibited growth of UCB derived CFU-GM. OS treatment increased the number of annexin-V CD34+ cells and NBT positive HL60 cells indicating that OS inhibition of CFU-GM and HL60 cell growth can be attributed to induction of apoptosis and differentiation. From these studies, it can be concluded that dd-PCR is an excellent tool for the discovery of novel genes expressed in human HSPCs. Characterisation of the proteins encoded by the novel genes ORP-3 and MERP-1 may reveal a regulatory role for these genes in haemopoiesis. Finally, investigations into the effects of OS on haemopoietic progenitor cells has revealed that OS are a new class of inhibitors of HSPC proliferation of potential relevance in vivo and in vitro.

Mitomycin C: a promising agent for the treatment of canine corneal scarring

Objective  To evaluate the safety and efficacy of mitomycin C (MMC) in prevention of canine corneal scarring.

Methods  With an in vitro approach using healthy canine corneas, cultures of primary canine corneal fibroblasts or myofibroblasts were generated. Primary canine corneal fibroblasts were obtained by growing corneal buttons in minimal essential medium supplemented with 10% fetal bovine serum. Canine corneal myofibroblasts were produced by growing cultures in serum-free medium containing transforming growth factor β1 (1 ng/mL). Trypan blue assay and phase-contrast microscopy were used to evaluate the toxicity of three doses of MMC (0.002%, 0.02% and 0.04%). Real-time PCR, immunoblot, and immunocytochemistry techniques were used to determine MMC efficacy to inhibit markers of canine corneal scarring.

Results  A single 2-min treatment of 0.02% or less MMC did not alter canine corneal fibroblast or keratocyte phenotype, viability, or growth. The 0.02% dose substantially reduced myofibroblast formation (up to 67%; P < 0.001), as measured by the change in RNA and protein expression of fibrosis biomarkers (α-smooth muscle actin and F-actin).

Conclusion  This in vitro study suggests that a single 2-min 0.02% MMC treatment to the canine corneal keratocytes is safe and may be useful in decreasing canine corneal fibrous metaplasia. In vivo studies are warranted.

The bone morphogenetic protein axis is a positive regulator of skeletal muscle mass

Although the canonical transforming growth factor β signaling pathway represses skeletal muscle growth and promotes muscle wasting, a role in muscle for the parallel bone morphogenetic protein (BMP) signaling pathway has not been defined. We report, for the first time, that the BMP pathway is a positive regulator of muscle mass. Increasing the expression of BMP7 or the activity of BMP receptors in muscles induced hypertrophy that was dependent on Smad1/5-mediated activation of mTOR signaling. In agreement, we observed that BMP signaling is augmented in models of muscle growth. Importantly, stimulation of BMP signaling is essential for conservation of muscle mass after disruption of the neuromuscular junction. Inhibiting the phosphorylation of Smad1/5 exacerbated denervation-induced muscle atrophy via an HDAC4-myogenin–dependent process, whereas increased BMP–Smad1/5 activity protected muscles from denervation-induced wasting. Our studies highlight a novel role for the BMP signaling pathway in promoting muscle growth and inhibiting muscle wasting, which may have significant implications for the development of therapeutics for neuromuscular disorders.

Effect of cell-surface components and metabolites of lactic acid bacteria and probiotic organisms on cytokine production and induction of CD25 expression in human peripheral mononuclear cells

In the current study, the relative contribution of cell-surface components (CSC) and cell-free supernatants (CFS) in the immuno-modulatory properties of 17 strains of probiotic and lactic acid bacteria (LAB) was assessed. The production of pro- and antiinflammatory cytokines including IL-2, IL-4, IL-10, IL-12 p70, IFN-γ, tumor necrosis factor-α (TNF-α), and transforming growth factor-β was measured at different time points after stimulation of buffy coat derived-peripheral blood mononuclear cells (PBMC) from healthy donors with CSC and CFS of probiotic and LAB. Results showed that CSC of probiotic and LAB strains induced production of T helper 1 and 2 type cytokines. Transforming growth factor-β was stimulated at highest concentrations, followed by IL-10 and TNF-α. The CFS of all tested bacterial strains induced PBMC for significantly high levels of IL-10 secretion compared with unstimulated cells, but the values were less than lipopolysaccharide-stimulated cells. Cytokines due to CFS stimulation showed declined concentration for IL-2, TNF-α, and IL-4, and complete disappearance of IL-12, IFN-γ, and transforming growth factor-β in the cultured medium at 96 h of incubation. Results of cytokine data demonstrate proinflammatory TNF-α immune responses are mainly directed through cell-surface structures of probiotic and LAB, but antiinflammatory immune responses are mediated both by metabolites and cell-surfaces of these bacteria. The induction of CD4(+)CD25(+) regulatory T cells after stimulation of PBMC with CSC and CFS of probiotic and LAB showed regulatory T cell activity appeared to be influenced both by the CSC and metabolites, but was principally triggered by cell surfaces of probiotic and LAB strains.

Novel targeting of PEGylated liposomes for codelivery of TGF-β1 siRNA and four antitubercular drugs to human macrophages for the treatment of mycobacterial infection: a quantitative proteomic study

Tuberculosis (TB) is still a major public health issue in developing countries, and its chemotherapy is compromised by poor drug compliance and severe side effects. This study aimed to synthesize and characterize new multimodal PEGylated liposomes encapsulated with clinically commonly used anti-TB drugs with linkage to small interfering RNA (siRNA) against transforming growth factor-β1 (TGF-β1). The novel NP-siRNA liposomes could target THP-1-derived human macrophages that were the host cells of mycobacterium infection. The biological effects of the NP-siRNA liposomes were evaluated on cell cycle distribution, apoptosis, autophagy, and the gene silencing efficiency of TGF-β1 siRNA in human macrophages. We also explored the proteomic responses to the newly synthesized NP-siRNA liposomes using the stable isotope labeling with amino acids in cell culture approach. The results showed that the multifunctional PEGylated liposomes were successfully synthesized and chemically characterized with a mean size of 265.1 nm. The novel NP-siRNA liposomes functionalized with the anti-TB drugs and TGF-β1 siRNA were endocytosed efficiently by human macrophages as visualized by transmission electron microscopy and scanning electron microscopy. Furthermore, the liposomes showed a low cytotoxicity toward human macrophages. There was no significant effect on cell cycle distribution and apoptosis in THP-1-derived macrophages after drug exposure at concentrations ranging from 2.5 to 62.5 μg/mL. Notably, there was a 6.4-fold increase in the autophagy of human macrophages when treated with the NP-siRNA liposomes at 62.5 μg/mL. In addition, the TGF-β1 and nuclear factor-κB expression levels were downregulated by the NP-siRNA liposomes in THP-1-derived macrophages. The Ingenuity Pathway Analysis data showed that there were over 40 signaling pathways involved in the proteomic responses to NP-siRNA liposome exposure in human macrophages, with 160 proteins mapped. The top five canonical signaling pathways were eukaryotic initiation factor 2 signaling, actin cytoskeleton signaling, remodeling of epithelial adherens junctions, epithelial adherens junction signaling, and Rho GDP-dissociation inhibitor signaling pathways. Collectively, the novel synthetic targeting liposomes represent a promising delivery system for anti-TB drugs to human macrophages with good selectivity and minimal cytotoxicity.

Cord blood monocyte-derived inflammatory cytokines suppress IL-2 and induce nonclassic "T(H)2-type" immunity associated with development of food allergy

Food allergy is a major health burden in early childhood. Infants who develop food allergy display a proinflammatory immune profile in cord blood, but how this is related to interleukin-4 (IL-4)/T helper 2 (T(H)2)-type immunity characteristic of allergy is unknown. In a general population-derived birth cohort, we found that in infants who developed food allergy, cord blood displayed a higher monocyte to CD4(+) T cell ratio and a lower proportion of natural regulatory T cell (nT(reg)) in relation to duration of labor. CD14(+) monocytes of food-allergic infants secreted higher amounts of inflammatory cytokines (IL-1β, IL-6, and tumor necrosis factor-α) in response to lipopolysaccharide. In the presence of the mucosal cytokine transforming growth factor-β, these inflammatory cytokines suppressed IL-2 expression by CD4(+) T cells. In the absence of IL-2, inflammatory cytokines decreased the number of activated nT(reg) and diverted the differentiation of both nT(reg) and naïve CD4(+) T cells toward an IL-4-expressing nonclassical TH2 phenotype. These findings provide a mechanistic explanation for susceptibility to food allergy in infants and suggest anti-inflammatory approaches to its prevention.

Epidermal growth factor-induced epithelio-mesenchymal transition in human breast carcinoma cells

PMC42-LA cells display an epithelial phenotype: the cells congregate into pavement epithelial sheets in which E-cadherin and beta-catenin are localized at cell-cell borders. They abundantly express cytokeratins, although 5% to 10% of the cells also express the mesenchymal marker vimentin. Stimulation of PMC42-LA cells with epidermal growth factor (EGF) leads to epithelio-mesenchymal transition-like changes including up-regulation of vimentin and down-regulation of E-cadherin. Vimentin expression is seen in virtually all cells, and this increase is abrogated by treatment of cells with an EGF receptor antagonist. The expression of the mesenchyme-associated extracellular matrix molecules fibronectin and chondroitin sulfate proteoglycan also increase in the presence of EGF. PMC42-LA cells adhere rapidly to collagen I, collagen IV, and laminin-1 substrates and markedly more slowly to fibronectin and vitronectin. EGF increases the speed of cell adhesion to most of these extracellular matrix molecules without altering the order of adhesive preference. EGF also caused a time-dependent increase in the motility of PMC42-LA cells, commensurate with the degree of vimentin staining. The increase in motility was at least partly chemokinetic, because it was evident both with and without chemoattractive stimuli. Although E-cadherin staining at cell-cell junctions disappeared in response to EGF, beta-catenin persisted at the cell periphery. Further analysis revealed that N-cadherin was present at the cell-cell junctions of untreated cells and that expression was increased after EGF treatment. N- and E-cadherin are not usually coexpressed in human carcinoma cell lines but can be coexpressed in embryonic tissues, and this may signify an epithelial cell population prone to epithelio-mesenchymal-like responses.

Epidermal growth factor-induced ovarian carcinoma cell migration is associated with JAK2/STAT3 signals and changes in the abundance and localization of α6β1 integrin

Peritoneal dissemination of ovarian carcinoma is mediated by epithelial–mesenchymal interconversions leading to the disruption of cell–cell contact and modulation of cell–extracellular matrix (ECM) interactions. The present study was designed to evaluate the effects of epidermal growth factor (EGF) as a modulator of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) signalling and changes in integrin expression during the process similar to EMT. A fibroblastic morphology with reduced intercellular cell contacts and increased cell motility was observed in ovarian cancer cell lines in response to EGF and was concomitant with the up regulation of EMT-associated N-cadherin and vimentin expression. These changes were accompanied by an increase in α2, α6 and β1 integrin subunits and activation of JAK2 and STAT3 signalling which was suppressed by a specific JAK2 inhibitor. Consistent with the suppression of STAT3 activity, N-cadherin and vimentin expression were abrogated and was coherent with the loss of cell motility and the expression of α6 and β1 integrin subunits. Neutralizing antibodies against α6 and β1 subunits inhibited cancer cell migration. A strong correlation between the expression of N-cadherin, vimentin and JAK2/STAT3 levels were detected in high-grade ovarian tumors and was consistent with the previously reported enhanced expression of α6 integrin subunit in advanced tumors [Ahmed N, Riley C, Oliva K, Rice G, Quinn M. Ascites induces modulation of α6β1 integrin and urokinase plasminogen activator receptor expression and associated functions in ovarian carcinoma. British Journal of Cancer 2005;92:1475–85]. Our data incorporating the clinical samples and the cancer cell lines is the first to demonstrate that JAK2/STAT3 pathway may be one of the downstream events in EMT-like process and α6β1 integrin-mediated signalling in ovarian carcinomas.

Tenocyte responses to mechanical loading in vivo : A role for local insulin-like growth factor 1 signaling in early tendinosis in rats

Objective
To investigate tenocyte regulatory events during the development of overuse supraspinatus tendinosis in rats.

Methods
Supraspinatus tendinosis was induced by running rats downhill at 1 km/hour for 1 hour a day. Tendons were harvested at 4, 8, 12, and 16 weeks and processed for brightfield, polarized light, or transmission electron microscopy. The development of tendinosis was assessed semiquantitatively using a modified Bonar histopathologic scale. Apoptosis and proliferation were examined using antibodies against fragmented DNA or proliferating cell nuclear antigen, respectively. Insulin-like growth factor 1 (IGF-1) expression was determined by computer-assisted quantification of immunohistochemical reaction. Local IGF-1 signaling was probed using antibodies to phosphorylated insulin receptor substrate 1 (IRS-1) and ERK-1/2.

Results
Tendinosis was present after 12 weeks of downhill running and was characterized by tenocyte rounding and proliferation as well as by glycosaminoglycan accumulation and collagen fragmentation. The proliferation index was elevated in CD90+ tenocytes in association with tendinosis and correlated with increased local IGF-1 expression by tenocytes and phosphorylation of IRS-1 and ERK-1/2. Both apoptosis and cellular inflammation were absent at all time points.

Conclusion
In this animal model, early tendinosis was associated with local stimulation of tenocytes rather than with extrinsic inflammation or apoptosis. Our data suggest a role for IGF-1 in the load-induced tenocyte responses during the pathogenesis of overuse tendon disorders.

Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1, a novel neuronal protein that regulates energy balance

To identify genes involved in the central regulation of energy balance, we compared hypothalamic mRNA from lean and obese Psammomys obesus, a polygenic model of obesity, using differential display PCR. One mRNA transcript was observed to be elevated in obese, and obese diabetic, P. obesus compared with lean animals and was subsequently found to be increased 4-fold in the hypothalamus of lethal yellow agouti (Ay/a) mice, a murine model of obesity and diabetes. Intracerebroventricular infusion of antisense oligonucleotide targeted to this transcript selectively suppressed its hypothalamic mRNA levels and resulted in loss of body weight in both P. obesus and Sprague Dawley rats. Reductions in body weight were mediated by profoundly reduced food intake without a concomitant reduction in metabolic rate. Yeast two-hybrid screening, and confirmation in mammalian cells by bioluminescence resonance energy transfer analysis, demonstrated that the protein it encodes interacts with endophilins, mediators of synaptic vesicle recycling and receptor endocytosis in the brain. We therefore named this transcript Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1 (SGIP1). SGIP1 encodes a large proline-rich protein that is expressed predominantly in the brain and is highly conserved between species. Together these data suggest that SGIP1 is an important and novel member of the group of neuronal molecules required for the regulation of energy homeostasis.

Fibroblast growth factor-2 over-rides insulin-like growth factor-I induced proliferation and cell survival in human neuroblastoma cells

The insulin-like growth factor (IGF) system is a key regulator of cell growth, survival and differentiation, and these functions are co-modulated by other growth factors including fibroblast growth factor-2 (FGF-2). To investigate IGF/FGF interactions in neuronal cells, we employed neuroblastoma cells (SK-N-MC). In serum free conditions proliferation of the SK-N-MC cells was promoted by IGF-I (25 ng/ml), but blunted by FGF-2 (50 ng/ml). IGF-I-induced proliferation was abolished in the presence of FGF-2 even when IGF-I was used at 100 ng/ml. In addition to our previously described FGF-2 induced proteolytic cleavage of IGFBP-2, we found that FGF-2 increased IGFBP-6 levels in conditioned medium (CM) without affecting IGFBP-6 mRNA abundance. Modulation of IGFBP-2 and -6 levels were not significant mechanisms involved in the blockade of IGF-I action since the potent IGF-I analogues [QAYL]IGF-I and des(1-3)IGF-I (minimal IGFBP affinity) were unable to overcome FGF-2 inhibition of cell proliferation. FGF-2 treated cells showed morphological differentiation expressing the TUJ1 neuronal marker while cells treated with IGF-I alone showed no morphological change. When IGF-I was combined with FGF-2, however, cell morphology was indistinguishable from that seen with FGF-2 alone. FGF-2 inhibited proliferation and enhanced differentiation was also associated with a 70% increase in cell death. Although IGF-I alone was potently anti-apoptotic (60% decreased), IGF-I was unable to prevent apoptosis when administrated in combination with FGF-2. Gene-array analysis confirmed FGF-2 activation of the intrinsic and extrinsic apoptotic pathways and blockade of IGF anti-apoptotic signaling. FGF-2, directly and indirectly, overcomes the proliferative and anti-apoptotic activity of IGF-I by complex mechanisms, including enhancement of differentiation and apoptotic pathways, and inhibition of IGF-I induced anti-apoptotic signalling. Modulation of IGF binding protein abundance by FGF-2 does not play a significant role in inhibition of IGF-I induced mitogenesis.

Expression of the disintegrin metalloprotease ADAM-10 in prostate cancer and its regulation by dihydrotestosterone, insulin like growth factor -1 and epidermal growth factor in the prostate cell model LNCaP

Purpose: The disintegrin metalloprotease ADAM-10 is a multidomain metalloprotease that is potentially significant in tumor progression due to its extracellular matrix-degrading properties. Previously, ADAM-10 mRNA was detected in prostate cancer (PCa) cell lines; however, the presence of ADAM-10 protein and its cellular localization, regulation, and role have yet to be described. We hypothesized that ADAM-10 mRNA and protein may be regulated by growth factors such as 5α-dihydrotestosterone, insulin-like growth factor I, and epidermal growth factor, known modulators of PCa cell growth and invasion.

Experimental Design: ADAM-10 expression was analyzed by in situ hybridization and immunohistochemistry in prostate tissues obtained from 23 patients with prostate disease. ADAM-10 regulation was assessed using quantitative reverse transcription-PCR and Western blot analysis in the PCa cell line LNCaP.

Results: ADAM-10 expression was localized to the secretory cells of prostate glands, with additional basal cell expression in benign glands. ADAM-10 protein was predominantly membrane bound in benign glands but showed marked nuclear localization in cancer glands. By Western blot, the 100-kDa proform and the 60-kDa active form of ADAM-10 were synergistically up-regulated in LNCaP cells treated with insulin-like growth factor I plus 5α-dihydrotestosterone. Epidermal growth factor also up-regulated both ADAM-10 mRNA and protein.

Conclusions: This study describes for the first time the expression, regulation, and cellular localization of ADAM-10 protein in PCa. The regulation and membrane localization of ADAM-10 support our hypothesis that ADAM-10 has a role in extracellular matrix maintenance and cell invasion, although the potential role of nuclear ADAM-10 is not yet known.

Effect of insulin-like growth factor I on HIV type 1 long terminal repeat-driven chloramphenicol acetyltransferase expression

In this study, we have investigated the ability of insulin-like growth factor I (IGF-I) to inhibit HIV long terminal repeat (LTR)-driven gene expression. Using COS 7 cells cotransfected with tat and an HIV LTR linked to a chloramphenicol acetyltransferase (CAT) reporter, we observed that physiological levels of IGF-I (10-9 M) significantly inhibited CAT expression in a concentration- and time-dependent manner. IGF-I did not inhibit C AT expression in COS 7 cells transfected with pSVCAT, and did not affect CAT expression in the absence of cotransfection with tat . Transfection of HIV-1 proviral DNA into COS 7 cells +/- IGF-I resulted in a significant decrease ( p 0.05) in infectious virion production. Both IGF-I and Ro24-7429 inhibited LTR-driven C AT expression, while TNF- alpha -enhanced CAT expression was not affected by IGF-I. On the other hand, a plasmid encoding parathyroid hormone-related peptide exhibited dramatic additivity of inhibition of CAT expression in COS 7 cells. Finally, we show that in Jurkat or U937 cells cotransfected with HIVLTRCAT/tat, IGF-I significantly inhibited CAT expression. Further, interleukin 4 showed in U937 cells inhibition of CAT expression that was not additive to IGF-I induced inhibition. Our data demonstrate that IGF-I can specifically inhibit HIVLTRCAT expression. This inhibition may occur at the level of the tat /TAR interaction. Finally, this IGF-I effect is seen in target cell lines and similar paths of inhibition may be involved in the various cell types employed.

Effect of vascular endothelial growth factor upregulation on retinal gene expression in the Kimba mouse

Background:  The Kimba mouse carries a human vascular endothelial growth factor transgene causing retinal neovascularisation similar to that seen in diabetic retinopathy. Here, we examine the relationship between differential gene expression induced by vascular endothelial growth factor overexpression and the architectural changes that occur in the retinae of these mice.

Methods:  Retinal gene expression changes in juvenile and adult Kimba mice were assayed by microarray and compared with age-matched wild-type littermates. Transcription of selected genes was validated by quantitative real-time polymerase chain reaction. Protein translation was determined using immunohistochemistry and enzyme-linked immunosorbent assay.

Results:  Semaphorin 3C was upregulated, and nuclear receptor subfamily 2, group 3, member 3 (Nr2e3) was downregulated in juvenile Kimba mice. Betacellulin and endothelin 2 were upregulated in adults. Semaphorin 3C colocalized with glial fibrillary acidic protein in Müller cells of Kimba retinae at greater signal intensities than in wild type. Endothelin 2 colocalised to Müller cell end feet and extended into the outer limiting membrane. Endothelin receptor type B staining was most pronounced in the inner nuclear layer, the region containing Müller cell somata.

Conclusions:  An early spike in vascular endothelial growth factor induced significant long-term retinal neovascularisation associated with changes to the retinal ganglion, photoreceptor and Müller cells. Overexpression of vascular endothelial growth factor led to dysregulation of photoreceptor metabolism through differential expression of Nr2e3, endothelin 2, betacellulin and semaphorin 3C. Alterations in the expression of these genes may therefore play key roles in the pathological mechanisms that result from retinal neovascularisation.