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James F Collins

Publications and source records attributed to James F Collins.

At least 19 recordsLinked to original sources
PubMed2006

Gene chip analyses reveal differential genetic responses to iron deficiency in rat duodenum and jejunum.

Previous studies revealed novel genetic changes in the duodenal mucosa of iron-deprived rats during postnatal development. These observations are now extended to compare the genetic response to iron deficiency in the duodenum versus jejunum of 12-wk-old rats. cRNA samples were prepared from the duodenal and jejunal mucosa of three groups each of control and iron-deficient rats and hybridized with RAE 230A and 230B gene chips (Affymetrix). Stringent data reduction strategies were employed. Results showed that several genes were similarly induced in both gut segments, including DMT1, Dcytb, transferrin receptor 1, heme oxygenase 1, metallothionein, the Menkes copper ATPase (ATP7A), tripartitie motif protein 27, and the sodium-dependent vitamin C transporter. However, a subset of genes showed regulation in only one or the other gut segment. In duodenum only, gastrokine 1, trefoil factor 1 and claudin 2 were induced by iron-deficiency. Other genes previously identified were only regulated in the duodenum. Overall, these studies demonstrate similarities and distinct differences in the genetic response to iron deprivation in the duodenum versus jejunum and provide evidence that more distal gut segments also may play a role in increasing iron absorption in iron-deficiency anemia.

Animals↗
PubMed2005

Development of a chronic inhalation reference level for respirable crystalline silica.

Chronic inhalation exposure of workers to crystalline silica can result in silicosis. The general public can also be exposed to lower levels of crystalline silica from quarries, sand blasting, and entrained fines particles from surface soil. We have derived an inhalation chronic reference exposure level for silica, a level below which no adverse effects due to prolonged exposure would be expected in the general public. Incidence of silicosis and silica exposure data from a cohort of 2235 white South African gold miners yielded a reference level of 3 microg/m3) for respirable silica (particle size as defined occupationally) using a benchmark concentration approach. Data from cohorts of American gold miners, Chinese tin miners, diatomaceous earth workers, and black South African gold miners yielded similar results with a range of 3-10 microg/m3. Strengths of the chronic reference exposure level include the availability of several large long-term studies of inhalation in workers at varying exposure concentrations, adequate histopathological and radiologic analysis, adequate follow-up of exposed workers, a dose-response effect in several studies, observation of a No Observed Adverse Effect Level in the key study, and the power of the key study to detect a small effect. Uncertainties include the general underestimation of silicosis by radiography alone and the uncertainties in exposure estimation.

China↗
PubMed2005

Menkes Copper ATPase (Atp7a) is a novel metal-responsive gene in rat duodenum, and immunoreactive protein is present on brush-border and basolateral membrane domains.

We previously noted strong induction of genes related to intestinal copper homeostasis (Menkes Copper ATPase (Atp7a) and metallothionein) in the duodenal epithelium of iron-deficient rats across several stages of postnatal development (Collins, J. F., Franck, C. A., Kowdley, K. V., and Ghishan, F. K. (2005) Am. J. Physiol., 288, G964-G971). We now report significant copper loading in the livers and intestines of iron-deficient rats. These findings are consistent with the hypothesis that there is increased intestinal copper transport during iron deficiency. We additionally found that hepatic Atp7b gene expression does not change with iron deficiency, suggesting that liver copper excretion is not altered. We have developed polyclonal antibodies against rat ATP7A, and we demonstrate the specificity of the immunogenic reaction. We show that the ATP7A protein is present on apical domains of duodenal enterocytes in control rats and on brush-border and basolateral membrane domains in iron-deprived rats. This localization is surprising, as previous in vitro studies have suggested that ATP7A traffics between the trans-Golgi network and the basolateral membrane. We further demonstrate that ATP7A protein levels are dramatically increased in brush-border and basolateral membrane vesicles isolated from iron-deficient rats. Other experiments show that iron refeeding partially corrects the hematological abnormalities seen in iron-deficient rats but that it does not ameliorate ATP7A protein induction, suggesting that Atp7a does not respond to intracellular iron levels. We conclude that ATP7A is involved in copper loading observed during iron deficiency and that increased intestinal copper transport is of physiological relevance, as copper plays important roles in overall body iron homeostasis.

Adenosine Triphosphatases↗
PubMed2005

1alpha,25-Dihydroxyvitamin D3 upregulates FGF23 gene expression in bone: the final link in a renal-gastrointestinal-skeletal axis that controls phosphate transport.

Fibroblast growth factor (FGF)23 is a phosphaturic hormone that decreases circulating 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and elicits hypophosphatemia, both of which contribute to rickets/osteomalacia. It has been shown recently that serum FGF23 increases after treatment with renal 1,25(OH)(2)D(3) hormone, suggesting that 1,25(OH)(2)D(3) negatively feedback controls its levels by inducing FGF23. To establish the tissue of origin and the molecular mechanism by which 1,25(OH)(2)D(3) increases circulating FGF23, we administered 1,25(OH)(2)D(3) to C57BL/6 mice. Within 24 h, these mice displayed a dramatic elevation in serum immunoreactive FGF23, and the expression of FGF23 mRNA in bone was significantly upregulated by 1,25(OH)(2)D(3), but there was no effect in several other tissues. Furthermore, we treated rat UMR-106 osteoblast-like cells with 1,25(OH)(2)D(3), and real-time PCR analysis revealed a dose- and time-dependent stimulation of FGF23 mRNA concentrations. The maximum increase in FGF23 mRNA was 1,024-fold at 10(-7) M 1,25(OH)(2)D(3) after 24-h treatment, but statistically significant differences were observed as early as 4 h after 1,25(OH)(2)D(3) treatment. In addition, using cotreatment with actinomycin D or cycloheximide, we observed that 1,25(OH)(2)D(3) regulation of FGF23 gene expression occurs at the transcriptional level, likely via the nuclear vitamin D receptor, and is dependent on synthesis of an intermediary transfactor. These results indicate that bone is a major site of FGF23 expression and source of circulating FGF23 after 1,25(OH)(2)D(3) administration or physiological upregulation. Our data also establish FGF23 induction by 1,25(OH)(2)D(3) in osteoblasts as a feedback loop between these two hormones that completes a kidney-intestine-bone axis that mediates phosphate homeostasis.

Animals↗
PubMed2005

Identification of differentially expressed genes in response to dietary iron deprivation in rat duodenum.

We sought to identify novel genes involved in intestinal iron absorption by inducing iron deficiency in rats during postnatal development from the suckling period through adulthood. We then performed comparative gene chip analyses (RAE230A and RAE230B chips; Affymetrix) with cRNA derived from duodenal mucosa. Real-time PCR was used to confirm changes in gene expression. Genes encoding the apical iron transport-related proteins [divalent metal transporter 1 (DMT1) and duodenal cytochrome b] were strongly induced at all ages studied, whereas increases in mRNA encoding the basolateral proteins iron-regulated gene 1 and hephaestin were observed only by real-time PCR. In addition, transferrin receptor 1 and heme oxygenase 1 were induced. We also identified induction of novel genes not previously associated with intestinal iron transport. The Menkes copper ATPase (ATP7a) and metallothionein were strongly induced at all ages studied, suggesting increased copper absorption by enterocytes during iron deficiency. We also found significantly increased liver copper levels in 7- to 12-wk-old iron-deficient rats. Also upregulated at most ages examined were the sodium-dependent vitamin C transporter, tripartite motif protein 27, aquaporin 4, lipocalin-interacting membrane receptor, and the breast cancer-resistance protein (ABCG2). Some genes also showed decreased expression with iron deprivation, including several membrane transporters, metabolic enzymes, and genes involved in the oxidative stress response. We speculate that dietary iron deprivation leads to increased intestinal copper absorption via DMT1 on the brush-border membrane and the Menkes copper ATPase on the basolateral membrane. These findings may thus explain copper loading in the iron-deficient state. We also demonstrate that many other novel genes may be differentially regulated in the setting of iron deprivation.

Animals↗
PubMed2004

Effects of angiotensin II on NaPi-IIa co-transporter expression and activity in rat renal cortex.

The kidney plays a major role in reabsorption of phosphate with the majority occurring in the proximal tubule (PT). The type IIa sodium-phosphate co-transporter (NaPi-IIa) is the main player in PT. The purpose of current study was to determine the effect of angiotensin II (A-II) on membrane expression of NaPi-IIa in the rat renal cortex. A-II (500 ng/kg/min) was chronically infused into the Sprague-Dawley rats by miniosmotic pump for 7 days. The arterial pressure and circulating plasma A-II level along with urine output were markedly increased in A-II rats. There was diuresis but no natriuresis. The phosphate excretion increased sevenfold on day 4 and 5.7-fold on day 7. There was no change in Na-dependent Pi uptake in brush-border membrane (BBM) vesicles between A-II-treated group and control on day 4, however, there was a 43% increase on day 7. Western blot analysis of NaPi-IIa protein abundance showed a parallel pattern: no change after 4 days of treatment and a 48% increase after 7 days of treatment. However, Northern blot analysis of cortical RNA showed no change in NaPi-IIa mRNA abundance on day 7. A-II stimulation of Na/Pi co-transport activity is a result of increases in the expression of BBM NaPi-IIa protein level and that stimulation is most likely mediated by posttranscriptional mechanisms.

Angiotensin II↗
PubMed2004

NF1 transcriptional factor(s) is required for basal promoter activation of the human intestinal NaPi-IIb cotransporter gene.

The human intestinal type IIb Na+-P(i) cotransporter (hNaPi-IIb) gene promoter lacks a TATA box and has a high GC content in the 5'-flanking region. To understand the mechanism of hNaPi-IIb gene transcription, the current study was performed to characterize the minimal promoter region and transcriptional factor(s) necessary to activate gene expression in human intestinal cells (Caco-2). With the use of progressively shorter promoter constructs, a minimal promoter extending from bp -58 to +15 was identified and shown to direct high levels of hNaPi-IIb cotransporter expression in Caco-2 cells. Gel mobility shift assays (GMSAs) indicated that two regions could be bound by nuclear proteins from Caco-2 cells: region A at bp -26/-23 and region B at bp -44/-35. The introduction of mutations in region A abolished promoter activity, whereas mutations in region B had no effect. Deletion mutants of the same regions showed identical results. Furthermore, DNase I footprinting experiments confirmed the observation made by GMSAs. Additional studies, which used a specific nuclear factor 1 (NF1) antiserum, demonstrated that NF1 protein(s) binds to the minimal promoter at region A. These results indicated that the NF1 protein(s) is required to activate the basal transcription of hNaPi-IIb gene under normal growth conditions. This study has thus identified a new target gene in the small intestinal epithelium that is directly regulated by NF1 transcriptional factor(s).

Base Sequence↗
PubMed2004

1,25-dihydroxyvitamin D3 down-regulation of PHEX gene expression is mediated by apparent repression of a 110 kDa transfactor that binds to a polyadenine element in the promoter.

The PHEX gene encodes an endopeptidase expressed in osteoblasts that inactivates an uncharacterized peptide hormone, phosphatonin, which suppresses bone mineralization as well as renal phosphate reabsorption and vitamin D bioactivation. We demonstrate that 1alpha-25-dihydroxyvitamin D (1,25(OH)2D3), the, active renal vitamin D metabolite, decreases PHEX mRNA in the rat osteoblastic cell line, UMR-106, as well as in mouse calvaria. Promoter/reporter construct analysis of the murine PHEX gene in transfected UMR-106 cells localized the repressive effect of 1,25(OH)2D3 to the -133 to -74 bp region, and gel mobility shift experiments revealed that 1,25(OH)2D3 treatment of the cells diminished the binding of a nuclear protein(s) to a stretch of 17 adenines from bp -116 to -100 in the proximal PHEX promoter. Either overexpression of a dominant-negative vitamin D receptor (VDR) or deletion of this sequence of 17 A-T base pairs abolished the repressive effect of 1,25(OH)2D3 by attenuating basal promoter activity, indicating that this region mediates the 1,25(OH)2D3 response and is involved in basal transcription. South-western blot analysis and DNA affinity purification show that an unidentified 110 kDa nuclear protein binds to the poly(A) element. Because 1,25(OH)2D3-liganded VDR neither binds to the polyadenine region of the PHEX promoter nor directly influences the association of the 110 kDa transfactor, we conclude that 1,25(OH)2D3 indirectly decreases PHEX expression via VDR-mediated repression (or modification) of this novel transactivator. Thus, we have identified a cis-element required for PHEX gene transcription that participates in negative feedback control of PHEX expression and thereby modulates the actions of phosphatonin.

Adenine↗
PubMed2004

Effect of angiotensin-II on renal Na+/H+ exchanger-NHE3 and NHE2.

The purpose of the present study was to determine the effect of angiotensin II (A-II) on membrane expression of Na+/H+ exchange isoforms NHE3 and NHE2 in the rat renal cortex. A-II (500 ng/kg per min) was chronically infused into the Sprague-Dawley rats by miniosmotic pump for 7 days. Arterial pressure and circulating plasma A-II level were significantly increased in A-II rats as compared to control rats. pH-dependent uptake of 22Na+ study in the presence of 50 microM HOE-694 revealed that Na+ uptake mediated by NHE3 was increased approximately 88% in the brush border membrane from renal cortex of A-II-treated rats. Western blotting showed that A-II increased NHE3 immunoreactive protein levels in the brush border membrane of the proximal tubules by 31%. Northern blotting revealed that A-II increased NHE3 mRNA abundance in the renal cortex by 42%. A-II treatment did not alter brush border NHE2 protein abundance in the renal proximal tubules. In conclusion, chronic A-II treatment increases NHE3-mediated Na+ uptake by stimulating NHE3 mRNA and protein content.

Angiotensin II↗
PubMed2003

Regulation of intestinal NaPi-IIb cotransporter gene expression by estrogen.

The current experiments were designed to study the effect of beta-estradiol on type IIb sodium-coupled phosphate (NaPi-IIb) cotransporter gene expression. Uptake studies with intestinal brush-border membrane vesicles (BBMV) showed that estrogen treatment increased sodium-dependent phosphate absorption by approximately 45% in rat intestine. Northern blot analysis indicated that NaPi-IIb mRNA expression was increased by approximately 50% after estrogen treatment. Western blot analysis also detected an increase in BBMV NaPi-IIb protein expression in estrogen-treated rats. In human intestinal Caco-2 cells, NaPi-IIb mRNA abundance was increased approximately 60% after estrogen treatment, and this increase could be abolished by inhibition of gene transcription. Transfection studies with human NaPi-IIb promoter reporter constructs showed that the promoter was responsive to estrogen treatment. These studies demonstrate for the first time that estrogen stimulates intestinal sodium-dependent phosphate absorption in female rats. This stimulation is associated with increased NaPi-IIb mRNA and protein expression. Thus the effect of estrogen on intestinal Pi absorption may be partially due to activation of NaPi-IIb gene transcription.

Animals↗
PubMed2003

The SLC20 family of proteins: dual functions as sodium-phosphate cotransporters and viral receptors.

The SLC20 family transport proteins were originally identified as retroviral receptors (called Glvr-1 and Ram-1). Since then, they have been shown to function as sodium-phosphate (Na/P(i)) cotransporters, and have subsequently been classified as type III Na/P(i) cotransporters (now called Pit-1 and Pit-2). The Pit cotransporters share approximately 60% sequence homology, they have a high affinity for P(i), they are electrogenic with a coupling stoichiometry of >1 Na(+) per P(i) ion cotransported, and are inhibited by alkaline pH and phosphonoformic acid (PFA). Pit-1 and Pit-2 expression and/or activity has also been shown to be regulated by P(i) deprivation in some, but not all cells and tissues examined. The Pit-1 and Pit-2 cotransporters are widely expressed, but cell-type specific expression has only been investigated in bone, kidney and intestine. Both proteins are likely expressed on the basolateral membranes of polarized epithelial cells, where they are likely involved in cellular P(i) homeostasis. The Pit-1 and Pit-2 gene promoters have been cloned and characterized. While the exact roles of the Pit cotransporters in different cell types has not been definitively determined, they may be involved in important physiological pathways in bone, aortic smooth muscle cells, parathyroid glands, kidney and intestine.

Animals↗
PubMed2003

Transcriptional regulation of the human NaPi-IIb cotransporter by EGF in Caco-2 cells involves c-myb.

The type IIb sodium-phosphate (NaP(i)-IIb) cotransporter mediates intestinal phosphate absorption. Previous work in our laboratory has shown that EGF inhibited NaP(i)-IIb cotransporter expression through transcriptional regulation. To understand this regulation, progressively shorter human NaP(i)-IIb promoter constructs were used to define the EGF response region, and gel mobility shift assays (GMSAs) were used to characterize DNA-protein interactions. Promoter analysis determined that the EGF response region was located between -784 and -729 base pair (bp) of the promoter. GMSAs and overexpression studies revealed an interaction between this promoter region and c-myb transcription factor. Inhibition of EGF receptor activation restored promoter function. Further studies suggested that MAPK, PKC, and/or PKA pathways are involved in this regulation. In conclusion, these studies suggest that EGF decreases human NaP(i)-IIb gene expression by modifying the c-myb protein such that it inhibits transcriptional activation. We further conclude that this downregulation of promoter function is mediated by EGF-activated PKC/PKA and MAPK pathways. This is the first study that demonstrates involvement of c-myb in the regulation of intestinal nutrient absorption.

Base Sequence↗
PubMed2002

Functional characterization of the human intestinal NaPi-IIb cotransporter in hamster fibroblasts and Xenopus oocytes.

The recently cloned NaPi-IIb cotransporter is an apical membrane protein that is involved in the absorption of phosphate in the intestine. To expedite functional and structural studies, the human intestinal NaPi-IIb cotransporter was stably expressed in hamster fibroblast (PS120) cells. The hNaPi-IIb cDNA stably transfected cells exhibited a 1.8-fold higher sodium-dependent phosphate uptake than vector DNA transfected cells, and had a K(m) for Pi of approximately 106 microM and a K(m) for Na(+) of approximately 34 mM. The hNaPi-IIb cotransporter was also expressed in Xenopus oocytes and it exhibited a K(m) for Pi of approximately 113 microM and a K(m) for Na(+) of approximately 65 mM. The hNaPi-IIb cotransporter expressed in both PS120 cells and oocytes was inhibited by high external pH. Furthermore, the phosphate uptake mediated by the hNaPi-IIb cotransporter was inhibited by 5 mM phosphonoformic acid (PFA), 1 mM arsenate and 100 nM phorbol myristate acetate (PMA). These results demonstrate that the human intestinal NaPi-IIb cotransporter is functional when expressed in hamster fibroblasts, and that this model system may be useful in the future to identify NaPi-IIb cotransporter-specific inhibitors.

Animals↗
PubMed2002

Transcriptional regulation of the rat NHE3 gene. Functional interactions between GATA-5 and Sp family transcription factors.

Expression of sodium-hydrogen exchanger isoform 3 (NHE3) in the intestinal and renal epithelium plays a critical role in sodium absorption and acid/base homeostasis. To decipher rat NHE3 gene regulation, its cis-acting regulatory elements and associated transcription factors were characterized by transient transfection of Caco-2, IEC-6, Qt6, and Drosophila SL2 cells. Deletion and mutational analyses demonstrated that the atypical TATA box located at bp -26/-31 was not necessary for promoter activity, and that a -20/+8-bp fragment represents a functional initiator. Within the 81-bp upstream region, three Sp transcription factor binding sites were critical because their mutation drastically reduced promoter activity. The roles of Sp1 and Sp3 were further demonstrated by electromobility shift assay and by transactivation of the NHE3 promoter in SL2 cells by forced expression of Sp1 and Sp3. Both of these transcription factors were found to act synergistically with GATA-5 bound to a GATA box in exon 1 (+20/+23 bp). These studies demonstrate that rat NHE3 promoter is initiator-driven and controlled mainly by Sp1 and Sp3, which functionally interact with GATA-5. This interaction represents a novel regulatory mechanism, which is likely to participate in a gradient of intestinal gene expression along the crypt-villus axis.

Adenocarcinoma↗
PubMed2002

Age-dependent regulation of rat intestinal type IIb sodium-phosphate cotransporter by 1,25-(OH)(2) vitamin D(3).

The current studies were designed to characterize type IIb sodium-inorganic phosphate (P(i)) cotransporter (NaP(i)-IIb) expression and to assess the effect of 1,25-(OH)(2) vitamin D(3) on NaP(i)-IIb gene expression during rat ontogeny. Sodium-dependent P(i) absorption by intestinal brush-border membrane vesicles (BBMVs) decreased with age, and NaP(i)-IIb gene expression also decreased proportionally with age. 1,25-(OH)(2) vitamin D(3) treatment increased intestinal BBMV P(i) absorption by approximately 2.5-fold in suckling rats and by approximately 2.1-fold in adult rats. 1,25-(OH)(2) vitamin D(3) treatment also increased NaP(i)-IIb mRNA abundance by approximately 2-fold in 14-day-old rats but had no effect on mRNA expression in adults. Furthermore, in rat intestinal epithelial (RIE) cells, 1,25-(OH)(2) vitamin D(3) increased NaP(i)-IIb mRNA abundance, an effect that was abolished by actinomycin D. Additionally, human NaP(i)-IIb gene promoter activity in transiently transfected RIE cells showed approximately 1.6-fold increase after 1,25-(OH)(2) vitamin D(3) treatment. In conclusion, we demonstrate that the age-related decrease in intestinal sodium-dependent P(i) absorption correlates with decreased NaP(i)-IIb mRNA expression. Our data also suggest that the effect of 1,25-(OH)(2) vitamin D(3) on NaP(i)-IIb expression is at least partially mediated by gene transcription in suckling rats.

Aging↗
PubMed2002

Glucocorticoid regulation and glycosylation of mouse intestinal type IIb Na-P(i) cotransporter during ontogeny.

We sought to characterize expression of an apically expressed intestinal Na-P(i) cotransporter (Na-P(i)-IIb) during mouse ontogeny and to assess the effects of methylprednisolone (MP) treatment. In control mice, Na-P(i) uptake by intestinal brush-border membrane vesicles was highest at 14 days of age, lower at 21 days, and further reduced at 8 wk and 8-9 mo of age. Na-P(i)-IIb mRNA and immunoreactive protein levels in 14-day-old animals were markedly higher than in older groups. MP treatment significantly decreased Na-P(i) uptake and Na-P(i)-IIb mRNA and protein expression in 14-day-old mice. Additionally, the size of the protein was smaller in 14-day-old mice. Deglycosylation of protein from 14-day-old and 8-wk-old animals with peptide N-glycosidase reduced the molecular weight to the predicted size. We conclude that intestinal Na-P(i) uptake and Na-P(i)-IIb expression are highest at 14 days and decrease with age. Furthermore, MP treatment reduced intestinal Na-P(i) uptake approximately threefold in 14-day-old mice and this reduction correlates with reduced Na-P(i)-IIb mRNA and protein expression. We also demonstrate that Na-P(i)-IIb is an N-linked glycoprotein and that glycosylation is age dependent.

Aging↗