Finian Martin

Suppression Subtractive Hybridization Identifies High Glucose Levels as a Stimulus for Expression of Connective Tissue Growth Factor and Other Genes in Human Mesangial Cells

Accumulation of mesangial matrix is a pivotal event in the pathophysiology of diabetic nephropathy. The molecular triggers for matrix production are still being defined. Here, suppression subtractive hybridization identified 15 genes differentially induced when primary human mesangial cells are exposed to high glucose (30 mm versus 5 mm) in vitro. These genes included (a) known regulators of mesangial cell activation in diabetic nephropathy (fibronectin, caldesmon, thrombospondin, and plasminogen activator inhibitor-1), (b) novel genes, and (c) known genes whose induction by high glucose has not been reported. Prominent among the latter were genes encoding cytoskeleton-associated proteins and connective tissue growth factor (CTGF), a modulator of fibroblast matrix production. In parallel experiments, elevated CTGF mRNA levels were demonstrated in glomeruli of rats with streptozotocin-induced diabetic nephropathy. Mannitol provoked less mesangial cell CTGF expression in vitro than high glucose, excluding hyperosmolality as the key stimulus. The addition of recombinant CTGF to cultured mesangial cells enhanced expression of extracellular matrix proteins. High glucose stimulated expression of transforming growth factor β1 (TGF-β1), and addition of TGF-β1 to mesangial cells triggered CTGF expression. CTGF expression induced by high glucose was partially suppressed by anti-TGF-β1 antibody and by the protein kinase C inhibitor GF 109203X. Together, these data suggest that 1) high glucose stimulates mesangial CTGF expression by TGFβ1-dependent and protein kinase C dependent pathways, and 2) CTGF may be a mediator of TGFβ1-driven matrix production within a diabetic milieu.

Extracellular BMP-antagonist regulation in development and disease: tied up in knots.

Developmental processes are regulated by the bone morphogenetic protein (BMP) family of secreted molecules. BMPs bind to serine/threonine kinase receptors and signal through the canonical Smad pathway and other intracellular effectors. Integral to the control of BMPs is a diverse group of secreted BMP antagonists that bind to BMPs and prevent engagement with their cognate receptors. Tight temporospatial regulation of both BMP and BMP-antagonist expression provides an exquisite control system for developing tissues. Additional facets of BMP-antagonist biology, such as crosstalk with Wnt and Sonic hedgehog signaling during development, have been revealed in recent years. In addition, previously unappreciated roles for the BMP antagonists in kidney fibrosis and cancer have been elucidated. This review provides a description of BMP-antagonist biology, together with highlights of recent novel insights into the role of these antagonists in development, signal transduction and human disease.

Biliary excretion and intestinal metabolism of progesterone and estrogens in man.

The biliary excretion and intestinal metabolism (including intestinal mucosa and small intestine and bowel) of progesterone and estrogens in humans are reviewed here along with presentation of experimental results from other mammalian systems. In general, the biliary excretion of estrogens and the enterohepatic circulation of estrogen metabolites are more extensive than for progesterone. These processes may be of greater physiological importance because of the possible reformation of biologically active estrogens in the intestine, which occurs 2 ways: 1) by hydrolysis of biliary estrogen conjugates and absorption of the unconjugated estrogens which may partly reach general circulation, and 2) by production of biologically active estrogens from neutral steroids or less active estrogens in the intestinal tract, followed by absorption. Here, the kidneys also play a significant role in estrogen metabolism and conjugation. The quantitative contribution of liver, kidneys, and intestine to estrogen metabolism cannot be accurately assessed at present, but the liver and intestines probably play the most significant role, followed by the kidneys. Progesterone (from orally administered doses) and its metabolites are extensively metabolized in the intestine to compounds with less progestational activity; hence, synthetic progestins may alter the intestinal flora which in turn may influence the plasma levels of these compounds. On these subjects more research in indicated.

New susceptibility loci associated with kidney disease in Type 1 diabetes

Diabetic kidney disease, or diabetic nephropathy (DN), is a major complication of diabetes and the leading cause of end-stage renal disease (ESRD) that requires dialysis treatment or kidney transplantation. In addition to the decrease in the quality of life, DN accounts for a large proportion of the excess mortality associated with type 1 diabetes (T1D). Whereas the degree of glycemia plays a pivotal role in DN, a subset of individuals with poorly controlled T1D do not develop DN. Furthermore, strong familial aggregation supports genetic susceptibility to DN. However, the genes and the molecular mechanisms behind the disease remain poorly understood, and current therapeutic strategies rarely result in reversal of DN. In the GEnetics of Nephropathy: an International Effort (GENIE) consortium, we have undertaken a meta-analysis of genome-wide association studies (GWAS) of T1D DN comprising ∼2.4 million single nucleotide polymorphisms (SNPs) imputed in 6,691 individuals. After additional genotyping of 41 top ranked SNPs representing 24 independent signals in 5,873 individuals, combined meta-analysis revealed association of two SNPs with ESRD: rs7583877 in the AFF3 gene (P = 1.2×10−8) and an intergenic SNP on chromosome 15q26 between the genes RGMA and MCTP2, rs12437854 (P = 2.0×10−9). Functional data suggest that AFF3 influences renal tubule fibrosis via the transforming growth factor-beta (TGF-β1) pathway. The strongest association with DN as a primary phenotype was seen for an intronic SNP in the ERBB4 gene (rs7588550, P = 2.1×10−7), a gene with type 2 diabetes DN differential expression and in the same intron as a variant with cis-eQTL expression of ERBB4. All these detected associations represent new signals in the pathogenesis of DN.

Increased claudin-4 expression is associated with poor prognosis and high tumour grade in breast cancer

The role of intercellular tight junctions in breast epithelial cells is traditionally thought to be in maintaining polarity and barrier function. However, claudin‐4, a tight junction protein, is overexpressed in breast tumour cells compared to normal epithelial cells, which generally corresponds to a loss in polarity. The aim of this study was to investigate the distribution and potential clinical value of claudin‐4 in breast cancer, and to evaluate its usefulness as a prognostic and predictive biomarker. Expression of claudin‐4 was initially examined by Western blot analysis in a cohort of 88 breast tumours, and was found to correlate positively with tumour grade and negatively with ER. Claudin‐4 expression was then evaluated by immunohistochemistry in a larger cohort of 299 tumours represented on a tissue microarray. Claudin‐4 expression correlated positively with tumour grade and Her2, and negatively with ER. High claudin‐4 expression was also associated with worse breast cancer‐specific survival (p = 0.003), recurrence‐free survival (p = 0.025) and overall survival (p = 0.034). Multivariate analysis revealed that claudin‐4 independently predicted survival in the entire cohort (HR 1.95; 95%CI 1.01–3.79; p = 0.047) and in the ER positive subgroup treated with adjuvant tamoxifen (HR 4.34; 95%CI 1.14–16.53; p = 0.032). This relationship between increased claudin‐4 expression and adverse outcome was validated at the mRNA level in a DNA microarray dataset of 295 breast tumours. We conclude that high levels of claudin‐4 protein are associated with adverse outcome in breast cancer patients, including the subgroup of patients treated with adjuvant tamoxifen.

Key signalling nodes in mammary gland development and cancer. Mitogen-activated protein kinase signalling in experimental models of breast cancer progression and in mammary gland development.

Seven classes of mitogen-activated protein kinase (MAPK) intracellular signalling cascades exist, four of which are implicated in breast disease and function in mammary epithelial cells. These are the extracellular regulated kinase (ERK)1/2 pathway, the ERK5 pathway, the p38 pathway and the c-Jun N-terminal kinase (JNK) pathway. In some forms of human breast cancer and in many experimental models of breast cancer progression, signalling through the ERK1/2 pathway, in particular, has been implicated as being important. We review the influence of ERK1/2 activity on the organised three-dimensional association of mammary epithelial cells, and in models of breast cancer cell invasion. We assess the importance of epidermal growth factor receptor family signalling through ERK1/2 in models of breast cancer progression and the influence of ERK1/2 on its substrate, the oestrogen receptor, in this context. In parallel, we consider the importance of these MAPK-centred signalling cascades during the cycle of mammary gland development. Although less extensively studied, we highlight the instances of signalling through the p38, JNK and ERK5 pathways involved in breast cancer progression and mammary gland development.

Lipoxin, leukotriene, and PDGF receptors cross-talk to regulate mesangial cell proliferation

The lipoxygenase‐derived leukotrienes (LTs) are important proinflammatory lipid mediators. Lipoxins (LXs), more recently described lipoxygenase products, modulate many proinflammatory actions of LTs and have impressive proresolution properties. Mesangial cell (MC) proliferation is a central event in the pathogenesis of glomerulonephritis. LTD4‐induced proliferation of mesangial cells is modulated by LXA4. Here, we demonstrate that LXA4 inhibits PDGF‐ and LTD4‐stimulated proliferation through modulation of platelet‐derived growth factor receptor β (PDGFRβ) activation. Specifically, we demonstrate that LTD4 transactivates the PDGFRβ, a process associated with c‐src recruitment and ras activation. We demonstrate expression of cysLT1 and cysLT2 receptors in MCs. LTD4‐induced c‐src activation was insensitive to pertussis toxin and the cysLT1 receptor antagonist Zafirlukast but was blocked by the nonselective antagonist Pobilukast. We show that LXA4 inhibits LTD4‐stimulated activation of the PDGFRβ and that LXA4 modulates PDGF‐BB‐stimulated tyrosine phosphorylation of the PDGFRβ and subsequent mitogenic events. Furthermore, expression of recombinant LXA4 receptor (ALXR) in CHOK1 cells was associated with an attenuation of serum‐stimulated proliferation. These data demonstrate that LXA4 receptor (ALXR) activation is accompanied by antimitogenic effects coupled with inactivation of growth factor receptors, highlighting the complex cross‐talk between G protein‐coupled receptors and receptor tyrosine kinases in an inflammatory milieu. These data elaborate on the profile of cell signaling events that underpin the anti‐inflammatory and proresolution bioactions of LX.

BMP signalling: agony and antagony in the family

Bone morphogenetic proteins (BMPs) are secreted extracellular matrix (ECM)-associated proteins that regulate a wide range of developmental processes, including limb and kidney formation. A critical element of BMP regulation is the presence of secreted antagonists that bind and inhibit BMP binding to their cognate Ser/Thr kinase receptors at the plasma membrane. Antagonists such as Noggin, Chordin, Gremlin (Grem1), and twisted gastrulation-1 (Twsg1) have been shown to inhibit BMP action in a range of different cell types and developmental stage-specific contexts. Here we review new developments in the field of BMP and BMP antagonist biology during mammalian development and suggest strategies for targeting these proteins in human disease.

YY1 and NF1 both activate the human p53 promoter by alternatively binding to a composite element, and YY1 and E1A cooperate to amplify p53 promoter activity.

A novel transcription factor binding element in the human p53 gene promoter has been characterized. It lies about 100 bp upstream of the major reported start site for human p53 gene transcription. On the basis of DNase I footprinting studies, electromobility shift assay patterns, sequence specificity of binding, the binding pattern of purified transcription factors, effects of specific antibodies, and methylation interference analysis we have identified the site as a composite element which can bind both YY1 and NF1 in an independent and mutually exclusive manner. The site is conserved in the human, rat, and mouse p53 promoters. The occupancy of the site varies in a tissue-specific manner. It binds principally YY1 in nuclear extracts of rat testis and spleen and NF1 in extracts of liver and prostate. This may facilitate tissue-specific control of p53 gene expression. When HeLa cells were transiently transfected with human p53 promoter-chloramphenicol acetyltransferase reporter constructs, a mutation in this composite element which disabled YY1 and NF1 binding caused a mean 64% reduction in basal p53 promoter activity. From mutations which selectively impaired YY1 or NF1 binding and the overexpression of YY1 or NF1 in HeLa cells we concluded that both YY1 and NF1 function as activators when bound to this site. In transient cotransfections E1A could induce the activity of the p53 promoter to a high level; 12S E1A was threefold as efficient as 13S E1A in this activity, and YY1 bound to the composite element was shown to mediate 55% of this induction. Overexpressed YY1 was shown to be able to synergistically activate the p53 promoter with E1A when not specifically bound to DNA. Deletion of an N-terminal domain of E1A, known to be required for direct E1A-YY1 interaction and E1A effects mediated through transcriptional activator p300, blocked the E1A induction of p53 promoter activity.

Steroid absorption and enterohepatic recycling.

A short review on steroid absorption and enterohepatic recycling in man with special emphasis on contraceptive and related steroids is presented. Some new experimental data on the intestinal metabolism of steroids is described and includes further observations on the effect of antimicrobial agents on steroid hormone metabolism. Evidence is presented that plasma levels of steroids may be influenced if the intestinal microflora is altered. Some formation of biologically active steroids, like estradiol, may occur in the intestinal tract and this may have both biological and pathological consequences and may be influenced by such factors as diet and sex. It is concluded that our knowledge of the intestinal and especially, the mucosal metabolism of steroids is scanty and further studies are needed to clarify the role of the intestine and enterohepatic circulation in determining the bioavailability of natural and synthetic steroids.