Physiological roles and regulation of mammalian sulfate transporters

All cells require inorganic sulfate for normal function. Sulfate is among the most important macronutrients; in cells and is the fourth most abundant anion in human plasma (300 muM). Sulfate is the major sulfur source in many organisms, and because it is a hydrophilic anion that cannot passively cross the lipid bilayer of cell membranes, all cells require a mechanism for sulfate influx and efflux to ensure an optimal supply of sulfate in the body. The class of proteins involved in moving sulfate into or out of cells is called sulfate transporters. To date, numerous sulfate transporters have been identified in tissues and cells from many origins. These include the renal sulfate transporters NaSi-1 and sat-1, the ubiquitously expressed diastrophic dysplasia sulfate transporter DTDST, the intestinal sulfate transporter DRA that is linked to congenital chloride diarrhea, and the erythrocyte anion exchanger AE1. These transporters have only been isolated in the last 10-15 years, and their physiological roles and contributions to body sulfate homeostasis are just now beginning to be determined. This review focuses on the structural and functional properties of mammalian sulfate transporters and highlights some of regulatory mechanisms that control their expression in vivo, under normal physiological and pathophysiological states.

The expression of plasminogen activator system in a rat model of periodontal wound healing

Background: The plasminogen activator system has been proposed to play a role in proteolytic degradation of extracellular matrices in tissue remodeling, including wound healing. The aim of this study was to elucidate the presence of components of the plasminogen activator system during different stages of periodontal wound healing. Methods: Periodontal wounds were created around the molars of adult rats and healing was followed for 28 days. Immunohistochemical analyses of the healing tissues and an analysis of the periodontal wound healing fluid by ELISA were carried out for the detection of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), and 2 plasminogen activator inhibitors (PAI-1 and PAI-2). Results: During the early stages (days 1 to 3) of periodontal wound healing, PAI-1 and PAI-2 were found to be closely associated with the deposition of a fibrin clot in the gingival sulcus. These components were strongly associated with the infiltrating inflammatory cells around the fibrin clot. During days 3 to 7, u-PA, PAI-1, and PAI-2 were associated with cells (particularly monocytes/macrophages, fibroblasts, and endothelial cells) in the newly formed granulation tissue. During days 7 to 14, a new attachment apparatus was formed during which PAI-1, PAI-2, and u-PA were localized in both periodontal ligament fibroblasts (PDL) and epithelial cells at sites where these cells were attaching to the root surface. In the periodontal wound healing fluid, the concentration for t-PA increased and peaked during the first week. PAI-2 had a similar expression to t-PA, but at a lower level over the entire wound-healing period. Conclusions: These findings indicate that the plasminogen activator system is involved in the entire process of periodontal wound healing, in particular with the formation of fibrin matrix on the root surface and its replacement by granulation tissue, as well as the subsequent formation of the attachment of soft tissue to the root surface during the later stages of wound repair.

EGF sensitizes cells to ionizing radiation by down-regulating protein mutated in ataxia-telangiectasia

Epidermal growth factor (EGF) has been reported to either sensitize or protect cells against ionizing radiation. We report here that EGF increases radiosensitivity in both human fibroblasts and lymphoblasts and down-regulates both ATM (mutated in ataxia-telangiectasia (A-T)) and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). No further radiosensitization was observed in A-T cells after pretreatment with EGF. The down-regulation of ATM occurs at the transcriptional level. Concomitant with the down-regulation of ATM, the DNA binding activity of the transcription factor Sp1 decreased. A causal relationship was established between these observations by demonstrating that up-regulation of Sp1 DNA binding activity by granulocyte/macrophage colony-stimulating factor rapidly reversed the EGF-induced decrease in ATM protein and restored radiosensitivity to normal levels. Failure to radiosensitize EGF-treated cells to the same extent as observed for A-T cells can be explained by induction of ATM protein and kinase activity with time post-irradiation. Although ionizing radiation damage to DNA rapidly activates ATM kinase and cell cycle checkpoints, we have provided evidence for the first time that alteration in the amount of ATM protein occurs in response to both EGF and radiation exposure. Taken together these data support complex control of ATM function that has important repercussions for targeting ATM to improve radiotherapeutic benefit.

UV-induced hyperphosphorylation of replication protein a depends on DNA replication and expression of ATM protein

Exposure to DNA-damaging agents triggers signal transduction pathways that are thought to play a role in maintenance of genomic stability. A key protein in the cellular processes of nucleotide excision repair, DNA recombination, and DNA double-strand break repair is the single-stranded DNA binding protein, RPA. We showed previously that the p34 subunit of RPA becomes hyperphosphorylated as a delayed response (4-8 h) to UV radiation (10-30 J/m(2)). Here we show that UV-induced RPA-p34 hyperphosphorylation depends on expression of ATM, the product of the gene mutated in the human genetic disorder ataxia telangiectasia (A-T). UV-induced RPA-p34 hyperphosphorylation was not observed in A-T cells, but this response was restored by ATM expression. Furthermore, purified ATM kinase phosphorylates the p34 subunit of RPA complex in vitro at many of the same sites that are phosphorylated in vivo after UV radiation. Induction of this DNA damage response was also dependent on DNA replication; inhibition of DNA replication by aphidicolin prevented induction of RPA-p34 hyperphosphorylation by UV radiation. We postulate that this pathway is triggered by the accumulation of aberrant DNA replication intermediates, resulting from DNA replication fork blockage by UV photoproducts. Further, we suggest that RPA-p34 is hyperphosphorylated as a participant in the recombinational postreplication repair of these replication products. Successful resolution of these replication intermediates reduces the accumulation of chromosomal aberrations that would otherwise occur as a consequence of UV radiation.

Epidermal growth factor sensitizes cells to ionizing radiation by down-regulating protein mutated in ataxia-telangiectasia

Epidermal growth factor (EGF) has been reported to either sensitize or protect cells against ionizing radiation. We report here that EGF increases radiosensitivity in both human fibroblasts and lymphoblasts and downregulates both ATM (mutated in ataxia-telangiectasia (A-T)) and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). No further radiosensitization was observed in A-T cells after pretreatment with EGF. The down-regulation of ATM occurs at the transcriptional level. Concomitant with the down-regulation of ATM, the DNA binding activity of the transcription factor Spl decreased. A causal relationship was established between these:observations by demonstrating that upregulation of Spl DNA binding activity by granulocyte/ macrophage colony-stimulating factor rapidly reversed the EGF-induced decrease in ATM protein and restored radiosensitivity to normal levels. Failure to radiosensitize EGF-treated cells to the same extent as observed for A-T cells ban be explained by induction of ATM protein and kinase activity with time post-irradiation, Although ionizing radiation damage to DNA rapidly activates ATM kinase and cell cycle checkpoints, we have provided evidence for the first time that alteration in the amount of ATM protein occurs in response to both EGF and radiation exposure. Taken together these data support complex control of ATM function that has important repercussions for targeting ATM to improve radiotherapeutic benefit.

Transcripts of the MHM region on the chicken Z chromosome accumulate as non-coding RNA in the nucleus of female cells adjacent to the DMRT1 locus

The male hypermethylated (MHM) region, located near the middle of the short arm of the Z chromosome of chickens, consists of approximately 210 tandem repeats of a BamHI 2.2-kb sequence unit. Cytosines of the CpG dinucleotides of this region are extensively methylated on the two Z chromosomes in the male but much less methylated on the single Z chromosome in the female. The state of methylation of the MHM region is established after fertilization by about the 1-day embryonic stage. The MHM region is transcribed only in the female from the particular strand into heterogeneous, high molecular-mass, non-coding RNA, which is accumulated at the site of transcription, adjacent to the DMRT1 locus, in the nucleus. The transcriptional silence of the MHM region in the male is most likely caused by the CpG methylation, since treatment of the male embryonic fibroblasts with 5-azacytidine results in hypo-methylation and active transcription of this region. In ZZW triploid chickens, MHM regions are hypomethylated and transcribed on the two Z chromosomes, whereas MHM regions are hypermethylated and transcriptionally inactive on the three Z chromosomes in ZZZ triploid chickens, suggesting a possible role of the W chromosome on the state of the MHM region.

Estrogen receptors in human preadipocytes

Estrogen influences regional adipose tissue distribution and the accompanying cardiovascular disease risk. To elucidate the mechanisms of this link further, we assessed whether human preadipocytes (PAs) expressed estrogen receptors (ERs) and whether there were any regional or gender differences in ER complement. Human PAs expressed the ER alpha gene but not ERP by reverse transcriptase-polymerase chain reaction, possessed ER alpha protein on Western blotting, and displayed specific 17 beta -estradiol (E-2) binding with calculated dissociation constants of 0.78 nM, 0.96 nM, and 1.19 nM and maximal binding capacities of 9.3 fmol/mg, 14.6 fmol/ mg, and 18.2 fmol/mg from three whole cell binding assays. There were no regional differences in ER alpha complement for males or females. There were no gender differences in ER alpha complement for subcutaneous or visceral samples. We conclude that ER alpha but not ERP is present in human PAs. This suggests that the effect of estrogen on adipose tissue deposition has a contribution from the direct effect of estrogen on human PAs via ER alpha.

A caveolin dominant negative mutant associates with lipid bodies and induces intracellular cholesterol imbalance

Recent studies have indicated a role for caveolin in regulating cholesterol-dependent signaling events. In the present study we have analyzed the role of caveolins in intracellular cholesterol cycling using a dominant negative caveolin mutant. The mutant caveolin protein, cav-3(DGV) specifically associates with the membrane surrounding large lipid droplets. These structures contain neutral lipids, and are accessed by caveolin 1-3 upon overexpression. Fluorescence, electron, and video microscopy observations are consistent with formation of the membrane-enclosed lipid rich structures by maturation of subdomains of the ER. The caveolin mutant causes the intracellular accumulation of free cholesterol (FC) in late endosomes, a decrease in surface cholesterol and a decrease in cholesterol efflux and synthesis. The amphiphile U18666A acts synergistically with cav(DGV) to increase intracellular accumulation of FC. Incubation of cells with oleic acid induces a significant accumulation of full-length caveolins in the enlarged lipid droplets. We conclude that caveolin can associate with the membrane surrounding lipid droplets and is a key component involved in intracellular cholesterol balance and lipid transport in fibroblasts.

Inhibition of lipid raft-dependent signaling by a dystrophy-associated mutant of caveolin-3

Specific point mutations in caveolin-3, a predominantly muscle-specific member of the caveolin family, have been implicated in limb-girdle muscular dystrophy and in rippling muscle disease. We examined the effect of these mutations on caveolin-3 localization and function. Using two independent assay systems, Raf activation in fibroblasts and neurite extension in PC12 cells, we show that one of the caveolin-3 point mutants, caveolin-3-C71W, specifically inhibits signaling by activated H-Ras but not by K-Ras. To gain insights into the effect of the mutant protein on H-Ras signaling, we examined the localization of the mutant proteins in fibroblastic cells and in differentiating myotubes. Unlike the previously characterized caveolin-3-DGV mutant, the inhibitory caveolin-3-C71W mutant reached the plasma membrane and colocalized with wild type caveolins. In BHK cells, caveolin-3-C71W associated with caveolae and in differentiating muscle cells with the developing T-tubule system. In contrast, the caveolin-3-P104L mutant accumulated in the Golgi complex and had no effect on H-Ras-mediated Raf activation. Inhibition by caveolin-3-C71W was rescued by cholesterol addition, suggesting that the mutant protein perturbs cholesterol-rich raft domains. Thus, we have demonstrated that a naturally occurring caveolin-3 mutation can inhibit signaling involving cholesterol-sensitive raft domains.

Isolation (from a basal cell carcinoma) of a functionally distinct fibroblast-like cell type that overexpresses Ptch

In this study we report on the isolation and characterization of a nonepithelial, nontumorigenic cell type (BCC1) derived from a basal cell carcinoma from a patient. The BCC1 cells share many characteristics with dermal fibroblasts, such as the expression of vimentin, lack of expression of cytokeratins, and insensitivity to agents that cause growth inhibition and differentiation of epithelial cells; however, significant differences between BCC1 cells and fibroblasts also exist. For example, BCC1 cells are stimulated to undergo DNA synthesis in response to interferon-gamma, whereas dermal fibroblasts are not. More over, BCC1 cells overexpress the basal cell carcinoma-specific genes ptch and ptch2 . These data indicate that basal cell carcinomas are associated with a functionally distinct population of fibroblast-like cells that overexpress known tumor-specific markers (ptch and ptch2 ).

Clinicopathological significance of the 'keloid-like' collagen and myxoid stroma in advanced rectal cancer

Aim: To establish the histological categorization of fibrotic stroma which reflects the biological behaviour of advanced rectal cancer. Methods and results: Six hundred and twenty-seven surgically resected cases of advanced rectal carcinoma were examined. We histologically categorized fibrotic stroma in the invasive frontal region into three groups: type A, multiple fine and mature fibres were stratified into layers: type B, broad bands of eosinophilic hyalinized collagen ('keloid-like' collagen) were intermingled: type C, myxoid stroma. Type A stroma was observed in 63% of patients, type B stroma in 25%, type C stroma in 12%.. The incidence of type A stroma decreased in accordance with Dukes stage (98% in Dukes A: 73% in B: 41%, in C1: 29% in C2) and conversely, there was an increase of C type (0%, in Dukes A; 4%, in B: 20% in C1: 54% in C2). Stroma type had a significant correlation with long-term survival (80% of 5-year survival in type A stroma: 54% in type B: 26% in type C). Based on multivariate analysis. it was found that the stromal pattern had independent prognostic value, together with nodal involvement. growth pattern. and lymphocyte infiltration. Conclusions: Tumour fibrotic stroma may play an important role as a regulator of neoplastic behaviour. Pathological categorization of the fibrotic stroma is helpful for predicting the prognostic outcome of patients with rectal carcinoma.

Interacting roles of myofibroblasts, apoptosis and fibrogenic growth factors in the pathogenesis of renal tubulo-interstitial fibrosis

The interrelationship between myofibroblasts and fibrogenic growth factors in the pathogenesis of renal fibrosis is poorly defined. A temporal and spatial analysis of myofibroblasts, their proliferation and death, and presence of transforming growth factor-beta1 (TGF-beta1) and platelet-derived growth factor-B (PDGF-B) was carried out in an established rodent model in which chronic renal scarring and fibrosis occurs after healed renal papillary necrosis (RPN), similar to that seen with analgesic nephropathy. Treated and control groups (N = 6 and 4, respectively) were compared at 2, 4, 8 and 12 weeks. A positive relationship was found between presence of tubulo-interstitial myofibroblasts and development of fibrosis. Apoptotic myofibroblasts were identified in the interstitium and their incidence peaked 2 weeks after treatment. Levels of interstitial cell apoptosis and fibrosis were negatively correlated over time (r = -0.57, p < 0.01 ), suggesting that as apoptosis progressively failed to limit myofibroblast numbers, fibrosis increased. In comparison with the diminishing apoptosis in the interstitium, the tubular epithelium had progressively increasing levels of apoptosis over time, indicative of developing atrophy of nephrons. TGF-beta1 protein expression had a close spatial and temporal association with fibrosis and myofibroblasts, whilst PDGF-B appeared to have a closer link with populations of other chronic inflammatory cells such as infiltrating lymphocytes. Peritubular myofibroblasts were often seen near apoptotic cells in the tubular epithelium, suggestive of a paracrine toxic effect of factor/s secreted by the myofibroblasts. In vitro , TGF-beta1 was found to be toxic to renal tubular epithelial cells. These findings suggest an interaction between myofibroblasts, their deletion by apoptosis, and the presence of the fibrogenic growth factor TGF-beta1 in renal fibrosis, whereby apoptotic deletion of myofibroblasts could act as a controlling factor in progression of fibrosis.

Intrinsic regional differences in androgen receptors and dihydrotestosterone metabolism in human preadipocytes

Androgens play an important role in regulating the central obesity that is a strong risk factor for cardiovascular disease and insulin resistance. This study confirms that androgen receptors are present in subcultured human preadipocytes, with androgen receptor gene expression and saturable specific dihydrotestosterone binding, dissociation constant 1.02 - 2.56 nM and maximal binding capacity 30.8 - 55.7 fmol/mg protein. There was an intrinsic regional difference in androgen receptor complement, with more androgen receptors in visceral than in subcutaneous preadipocytes. Dihydrotestosterone was metabolised by human preadipocytes, with more androstanediol produced by subcutaneous than visceral preadipocytes. While dihydrotestosterone metabolism was insufficient to explain the regional variation in androgen binding, both of these differences would reduce the androgen responsiveness of the subcutaneous preadipocytes compared with visceral preadipocytes. There were no gender differences in androgen binding or metabolism. While the direct effects of androgens on human PAS remain uncertain, these regional differences suggest that AR-mediated regulation of certain PA functions influences adipose tissue distribution.

Thermolysin activates equine lamellar hoof matrix metalloproteinases

Cultured equine lamellar hoof explants secrete the pro-enzymes matrix metalloproteinse-2 (MMP-2, 72 kDa) and MMP-2 (92 kDa). Untreated explants remained intact tested on a calibrated force transducer, but when treated with an NIMP activator, developed in-vitro laminitis, separating at the dermal-epidermal junction. Explants treated with the bacterial protease thermolysin separated dose-dependently; this was accompanied by activation of both MMP-2 and -9. Thermolysin-mediated NIP activation did not occur in a cell-free system and was not inhibited by the addition of the MMP inhibitor and batimastat. These findings suggest that thermolysin-mediated gelatinase activation is not dependent on membrane-bound matrix metalloproteinase (MT-MMP) activation, providing further evidence that bacteria can produce potent MMP activators that probably facilitate host invasion. (C) 2002 Harcourt Publishers Ltd.