Biomedical subjects

Madeline Murphy

Publications and source records attributed to Madeline Murphy.

14 recordsLinked to original sources

PubMed2006

Recapitulation of embryological programmes in renal fibrosis--the importance of epithelial cell plasticity and developmental genes.

Chronic fibrosis represents the final common pathway in progressive renal disease. Myofibroblasts deposit the constituents of renal scar, thus crippling renal function. It has recently emerged that an important source of these pivotal effector cells is the injured renal epithelium. This review concentrates on the process of epithelial-mesenchymal transition (EMT) and its regulation. The role of the developmental gene, gremlin, which is reactivated in adult renal disease, is the subject of particular focus. This member of the cysteine knot protein superfamily is critical to the process of nephrogenesis but quiescent in normal adult kidney. There is increasing evidence that gremlin expression reactivates in diabetic nephropathy, and in the diseased fibrotic kidney per se. Known to antagonize members of the bone morphogenic protein (BMP) family, gremlin may also act downstream of TGF-beta in induction of EMT. An increased understanding of the extracellular modulation of EMT and, in particular, of the gremlin-BMP axis may result in strategies that can halt or reverse the devastating progression of chronic renal fibrosis.

Animals↗

PubMed2006

Differential expression of connexin 43 in mouse mammary cells.

In this study we have employed suppressive subtractive hybridization (SSH) analysis to investigate differential gene expression in primary mouse mammary epithelial cells (PMMEC) cultured under mildly apoptotic/quiescent and differentiating conditions. Among a small group of genes whose expression was differentially regulated was connexin 43. In vitro, connexin 43 mRNA and protein were detectable in PMMEC cultured under proliferative or mildly apoptotic conditions. The level of connexin 43 mRNA expression in vivo was also investigated. High levels of expression were found to be associated with the periods of greatest glandular plasticity (pubertal expansion of the mammary tree, early pregnancy and during early involution). Thus, terminally differentiated cells in vivo and in vitro did not express connexin 43 mRNA suggesting that connexin 43 expression, and perhaps facilitated gap junction communication, is associated with undifferentiated progenitor cell populations.

Animals↗

PubMed2006

Heat shock-induced protection of renal proximal tubular epithelial cells from cold storage and rewarming injury.

Cold storage and reperfusion injury to transplanted kidneys contributes to increased incidence of delayed graft function and may have a negative impact on graft survival. This study examined the mechanisms by which previous heat shock protects against cell death in an in vitro model of kidney storage. Cold storage is mimicked by incubating human renal proximal tubular epithelial (HK-2) cells in University of Wisconsin solution at 4 degrees C with and without subsequent rewarming. Heat shock was induced by incubation of cells at 42 degrees C for 1 h. Altered protein expression was measured by Western blot, and cell viability and apoptosis were measured by propidium iodide DNA staining using flow cytometry. The specific role of heat-shock protein 70 (HSP-70) was determined both by siRNA knockdown and by stable overexpression approaches. Cold storage and rewarming-induced cell death was associated with decreased expression of HSP-70, HSP-90, HSP-27, and Bcl-2. Previous heat shock significantly reduced HK-2 cell death after cold storage and rewarming and was associated with the maintenance of HSP-70, HSP-27, and Bcl-2 protein levels. Blocking heat stress-induced HSP-70 with siRNA did not significantly block the protective effect of heat stress against cold storage and rewarming cell death; however, overexpression of HSP-70 protected HK-2 cells from this stress. It is concluded that previous heat shock protects HK-2 cells from cold storage and rewarming injury. siRNA inhibition of HSP-70 induction did not block the protective effect of heat shock, indicating that HSP-70 is not essential to the heat stress-induced protective effect reported in this study.

Blotting, Western↗

PubMed2005

TGF-beta1-induced EMT can occur independently of its proapoptotic effects and is aided by EGF receptor activation.

Apoptosis and epithelial-mesenchymal transdifferentiation (EMT) occur in stressed tubular epithelial cells and contribute to renal fibrosis. Transforming growth factor (TGF)-beta(1) promotes these responses and we examined whether the processes were interdependent in vitro. Direct (caspase inhibition) and indirect [epidermal growth factor (EGF) receptor stimulation] strategies were used to block apoptosis during TGF-beta(1) stimulation, and the subsequent effect on EMT was assessed. HK-2 cells were exposed to TGF-beta(1) with or without preincubation with ZVAD-FMK (pan-caspase inhibitor) or concomitant treatment with EGF plus or minus preincubation with LY-294002 (PI3-kinase inhibitor). Cells were then assessed for apoptosis and proliferation by flow cytometry, crystal violet assay, and Western blotting. Markers of EMT were assessed by microscopy, immunofluorescence, real-time RT-PCR, Western blotting, PAI-1 reporter assay, and collagen gel contraction assay. TGF-beta(1) caused apoptosis and priming for staurosporine-induced apoptosis. This was blocked by ZVAD-FMK. However, ZVAD-FMK did not prevent EMT following TGF-beta(1) treatment. EGF inhibited apoptosis and facilitated TGF-beta(1) induction of EMT by increasing proliferation and accentuating E-cadherin loss. Additionally, EGF significantly enhanced TGF-beta(1)-induced collagen I gel contraction. EGF increased Akt phosphorylation during EMT, and the prosurvival effect of this was confirmed using LY-294002, which reduced EGF-induced Akt phosphorylation and reversed its antiapoptotic and proproliferatory effects. TGF-beta(1) induces EMT independently of its proapoptotic effects. TGF-beta(1) and EGF together lead to EMT. EGF increases proliferation and resistance to apoptosis during EMT in a PI3-K Akt-dependent manner. In vivo, EGF receptor activation may assist in the selective survival of a transdifferentiated, profibrotic cell type.

Apoptosis↗

PubMed2005

Transforming growth factor-beta-regulated gene transcription and protein expression in human GFAP-negative lamina cribrosa cells.

Primary open-angle glaucoma (POAG) is a progressive optic neuropathy, which is a major cause of worldwide visual impairment and blindness. Pathological hallmarks of the glaucomatous optic nerve head (ONH) include retinal ganglion cell axon loss and extracellular matrix (ECM) remodeling of the lamina cribrosa layer. Transforming growth factor-beta (TGF-beta) is an important pro-fibrotic modulator of ECM metabolism, whose levels are elevated in human POAG lamina cribrosa tissue compared with non-glaucomatous controls. We hypothesize that in POAG, lamina cribrosa (LC) glial cells respond to elevated TGF-beta, producing a remodeled ONH ECM. Using Affymetrix microarrays, we report the first study examining the effect of TGF-beta1 on global gene expression profiles in glial fibrillary acidic acid (GFAP)-negative LC glial cells in vitro. Prominent among the differentially expressed genes were those with established fibrogenic potential, including CTGF, collagen I, elastin, thrombospondin, decorin, biglycan, and fibromodulin. Independent TaqMan and Sybr Green quantitative PCR analysis significantly validated genes involved in regulation of cell proliferation (platelet-derived growth factor [PDGF-alpha]), angiogenesis (vascular endothelial growth factor [VEGF]), ECM accumulation and degradation (CTGF, IL-11, and ADAMT-S5), and growth factor binding (ESM-1). Bioinformatic analysis of the ESM-1 promoter identified putative Smad and Runx transcription factor binding sites, and luciferase assays confirmed that TGF-beta1 drives transcription of the ESM-1 gene. TGF-beta1 induces expression and release of ECM components in LC cells, which may be important in regulating matrix remodeling in the lamina cribrosa. In disease states such as POAG, the LC cell may represent an important pro-fibrotic cell type and an attractive target for novel therapeutic strategies.

Cells, Cultured↗

PubMed2005

Expression of gremlin, a bone morphogenetic protein antagonist, in human diabetic nephropathy.

BACKGROUND: We report the induction of gremlin, a bone morphogenetic protein antagonist, in cultured human mesangial cells exposed to high glucose and transforming growth factor beta (TGF-beta) levels in vitro and kidneys from diabetic rats in vivo. METHODS: Gremlin expression was assessed in human diabetic nephropathy by means of in situ hybridization, immunohistochemistry, and real-time polymerase chain reaction and correlated with clinical and pathological indices of disease. RESULTS: Gremlin was not expressed in normal human adult kidneys. Conversely, abundant gremlin expression was observed in human diabetic nephropathy. Although some gremlin expression was observed in occasional glomeruli, gremlin expression was most prominent in areas of tubulointerstitial fibrosis, where it colocalized with TGF-beta expression. Gremlin messenger RNA levels correlated directly with renal dysfunction, determined by means of serum creatinine level, but not with proteinuria level. There was a strong correlation between gremlin expression and tubulointerstitial fibrosis score. CONCLUSION: In aggregate, these results indicate that the developmental gene gremlin reemerges in the context of tubulointerstitial fibrosis in diabetic nephropathy and suggests a role for TFG-beta as an inducer of gremlin expression in this context.

Bone Morphogenetic Proteins↗

PubMed2004

Analysis of differential maternal mRNA expression in developmentally competent and incompetent bovine two-cell embryos.

The main objective of this study was to identify mRNA transcripts associated with embryonic developmental competence. In cattle, mRNA transcripts, ribosomes, and proteins accumulated during the growth phase are drawn on to sustain maturation, fertilization, and the initial cell cycle divisions up to the 8- to 16-cell stage. Early cleaving mammalian zygotes are more likely to develop to the blastocyst stage than their later cleaving counterparts, thus reflecting the intrinsic quality of the oocytes from which they originated. We describe the combination of this well-established model for the retrospective determination of developmental competence in mammalian oocytes with a technique for wide screening of differential gene expression in different biological populations. Immature cumulus oocyte complexes were recovered from surface visible follicles on abattoir ovaries, washed, and submitted to routine in vitro maturation and fertilization. Two-cell embryos were removed from culture at 3-hr intervals from 24 to 42 hr post insemination (pi). Two populations of two-cell embryos were identified; those that cleaved early (before 27 hpi) and those that cleaved late (after 33 hpi). Suppressive subtractive hybridization was carried out on cDNA from the two populations, following which, differentially expressed amplicons were subcloned and sequenced. The sequences were submitted to the nonredundant and expressed sequence tag (EST) databases at NCBI using the BLAST algorithm. The differential expression of three selected candidate genes that were identified as putatively upregulated in the early cleaving zygotes were chosen for further investigations; histone H3, cyclin B1, and GDF-9B. Using quantitative real time PCR we have shown that histone H3A is significantly more abundant in embryos that cleave earliest.

Animals↗

PubMed2004

Association between variation in the actin-binding gene caldesmon and diabetic nephropathy in type 1 diabetes.

Dysfunction of the actin cytoskeleton is a key event in the pathogenesis of diabetic nephropathy. We previously reported that certain cytoskeletal genes are upregulated in mesangial cells exposed to a high extracellular glucose concentration. One such gene, caldesmon, lies on chromosome 7q35, a region linked to nephropathy in family studies, making it a candidate susceptibility gene for diabetic nephropathy. We screened all exons, untranslated regions, and a 5-kb region upstream of the gene for variation using denaturing high-performance liquid chromatography technology. An A>G single nucleotide polymorphism (SNP) at position -579 in the promoter region was associated with nephropathy in a case-control study using 393 type 1 diabetic patients from Northern Ireland (odds ratio [OR] 1.38, 95% CI 1.02-1.86, P = 0.03). A similar trend was found in an independent sample from a second center. When the sample groups were combined (n = 606), the association between the -579G allele and nephropathy remained significant (OR 1.35, 1.07-1.70, P = 0.01). The haplotype structure in the surrounding 7-kb region was determined. No single haplotype was more strongly associated with nephropathy than the -579A>G SNP. These results suggest a role for the caldesmon gene in susceptibility to diabetic nephropathy in type 1 diabetes.

Base Sequence↗

PubMed2003

Search for the bovine homolog of the murine ped gene and characterization of its messenger RNA expression during bovine preimplantation development.

In mice, a gene (Ped: preimplantation embryo development) that regulates preimplantation embryonic growth, including cleavage rate and embryo survivability, has been described. The objective of the current study was to identify the bovine homolog of the Ped gene and to characterize the mRNA expression pattern of this gene during bovine preimplantation embryo development. The NCBI GenBank/EBI expressed sequence tags (EST) databases were searched for bovine ESTs that were homologous to the murine Ped gene, and the resulting ESTs were aligned and assembled into a contiguous sequence. The homology of the sequence to the murine Ped gene was confirmed. Primers were designed for the sequence, and the mRNA expression pattern was characterized during bovine preimplantation embryo development in vivo and in vitro. In vitro-produced bovine zygotes were cultured either in vitro, in synthetic oviduct fluid, or in vivo in the ewe oviduct for 1-7 days and processed for quantitative real-time polymerase chain reaction (PCR). Transcript abundance increased at each stage of development. However, the expression levels were consistently higher in in vivo-cultured embryos at all stages, with in vivo-cultured embryos showing a 9-fold increase in relative transcript abundance during culture from the zygote to the blastocyst stage in contrast to just under a 4-fold increase during the same culture period in vitro. The mRNA expression pattern of the gene was investigated in early- and late-cleaving two-cell embryos collected at 25, 28, 32, and >or=36 h postinsemination (pi). Transcript relative abundance was highest in those embryos that had cleaved by 28 hpi and decreased almost 3-fold thereafter. In conclusion, we have identified a potential bovine homolog of the murine Ped gene. We have characterized the mRNA expression pattern of this gene during preimplantation embryo development in cattle and shown that a greater relative abundance of the gene transcript is associated with embryos of higher quality (in vivo cultured) and greater developmental potential (early cleaving).

Amino Acid Sequence↗

PubMed2003

Gremlin - a putative pathogenic player in progressive renal disease.

Progressive renal fibrosis is the end process of renal injury leading to kidney failure. Current therapies for chronic renal failure aim to slow this process but fail to halt its progression. As the mechanisms involved in glomerulosclerosis and tubulointerstitial fibrosis are unravelled, potential treatments for this growing clinical problem should emerge. Gremlin, a developmental regulator of bone morphogenetic proteins (BMPs), has recently been implicated in processes such as glomerulosclerosis, tubulointerstitial fibrosis and cellular hypertrophy, and may represent a novel therapeutic target in progressive renal diseases.

Animals↗

PubMed2002

The role of p42/44 MAPK and protein kinase B in connective tissue growth factor induced extracellular matrix protein production, cell migration, and actin cytoskeletal rearrangement in human mesangial cells.

Connective tissue growth factor (CTGF) is a member of an emerging family of immediate-early gene products that coordinate complex biological processes during differentiation and tissue repair. Here we describe the role of CTGF in integrin-mediated adhesive signaling and the production of extracellular matrix components in human mesangial cells. The addition of CTGF to primary mesangial cells induced fibronectin production, cell migration, and cytoskeletal rearrangement. These functional responses were associated with recruitment of Src and phosphorylation of p42/44 MAPK and protein kinase B. The inhibition of CTGF-induced p42/44 MAPK or phosphatidylinositol 3-kinase (PI3K)/protein kinase B pathway activities abrogated the induction of fibronectin expression. In addition, anti-beta(3) integrin antibodies attenuated the activation of both the p42/44 MAPK and protein kinase B and the increase in fibronectin levels. CTGF also induced mesangial cell migration via a beta(3) integrin-dependent mechanism that was similarly sensitive to the inhibition of the p42/44 MAPK and PI3K pathways, and it promoted the adhesion of the mesangial cells to type I collagen via up-regulation of alpha(1) integrin. Transient actin cytoskeletal disassembly was observed following treatment with the ligand over the course of a 24-h period. CTGF induced the loss of focal adhesions from the mesangial cell as evidenced by the loss of punctate vinculin. However, these processes are p42/44 MAPK and PI3K pathway-independent. Our data support the hypothesis that CTGF mediates a number of its biological effects by the induction of signaling processes via beta(3) integrin. However, others such as actin cytoskeleton disassembly are modulated in a beta(3) integrin/MAPK/PI3K-independent manner, indicating that CTGF is a complex pleiotropic factor with the potential to amplify primary pathophysiological responses.

Actins↗

PubMed2002

High glucose-altered gene expression in mesangial cells. Actin-regulatory protein gene expression is triggered by oxidative stress and cytoskeletal disassembly.

High extracellular glucose plays a pivotal role in the pathophysiology of diabetic nephropathy. Here we report 200 genes, identified using suppression-subtractive hybridization, that are differentially expressed when human mesangial cells are propagated in high ambient glucose in vitro. The major functional classes of genes identified included modulators and products of extracellular matrix protein metabolism, regulators of cell growth and turnover, and a cohort of actin cytoskeleton regulatory proteins. Actin cytoskeletal disassembly is a prominent feature of diabetic nephropathy. The induction of actin cytoskeleton regulatory gene expression by high glucose was attenuated by the inhibitor of reactive oxygen species generation, carbonyl cyanide m-chlorophenylhydrazone but not by the protein kinase C inhibitor GF 109203X and was not mimicked by the addition of transforming growth factor beta. Enhanced expression of actin cytoskeleton regulatory genes was also observed following disruption of the mesangial cell actin cytoskeleton by cytochalasin D. In aggregate, these results suggest that the induction of genes encoding actin cytoskeleton regulatory proteins (a) is a prominent component of the mesangial cell transcriptomic response in diabetic nephropathy and (b) is dependent on oxidative stress, is independent of protein kinase C and transforming growth factor-beta, and represents an adaptive response to actin cytoskeleton disassembly.

Actins↗

PubMed2002

Gremlin: an example of the re-emergence of developmental programmes in diabetic nephropathy.

The past two decades have yielded major advances in our understanding of the pathogenetic mechanisms that cause diabetic nephropathy. Of particular interest is the emerging paradigm of the recapitulation of developmental programmes within the diabetic kidney. Recently we have used the complementary techniques of suppression subtractive hybridization and Affymetrix GeneChips to assess changes in gene expression in human mesangial cells subjected to high ambient glucose concentrations and cyclic mechanical strain in vitro, the latter being models of hyperglycaemia and glomerular hypertension, respectively. In this review, we will focus on the potential role of one such differentially expressed gene, namely gremlin, in the pathogenesis of diabetic nephropathy. In the context of developmental nephrology, gremlin warrants special mention. Gremlin is a 184 amino acid protein and a member of the cysteine knot superfamily. The protein is highly conserved during evolution and is present in soluble and cell-associated forms. It belongs to a novel family of bone morphogenetic protein (BMP) antagonists that includes the head-inducing factor Cerberus and the tumour suppressor DAN. These proteins play important roles in limb development and neural crest cell differentiation. Evidence will be presented that mesangial cell gremlin expression is up-regulated by high ambient glucose, cyclic mechanical strain and transforming growth factor-beta (TGF-beta) and that gremlin may be an important modulator of mesangial cell proliferation and epithelial-mesenchymal transdifferentiation in a diabetic milieu.

Aging↗

PubMed2002

Gremlins: is this what renal fibrogenesis has come to?

Gremlin is a member of the differential screening-selected gene aberrative in the neuroblastoma (DAN) family of bone morphogenetic protein (BMP) antagonists. Gremlin influences diverse processes in growth, differentiation and development. Increased expression of gremlin has recently been demonstrated in several models of diabetic nephropathy. Gremlin arrests the cell cycle in mesangial cells and has also been shown to be upregulated in transdifferentiated renal proximal tubular cells. This review summarizes emerging evidence implicating gremlin in the pathophysiology of glomerulosclerosis and tubulointerstitial fibrosis. Gremlin is a potential novel therapeutic target in progressive renal diseases.

Animals↗