Biomedical subjects

Mark Donowitz

Publications and source records attributed to Mark Donowitz.

10 recordsLinked to original sources

PubMed2026

Aldosterone suppresses Na+/H+ exchanger-3 expression through miR-204-5P-mediated posttranscriptional regulation in distal colon.

Na+/H+ exchanger-3 (NHE3) is a major mediator of electroneutral NaCl absorption in the intestine and colon. In the distal colon, chronic aldosterone exposure suppresses NHE3 expression, but the molecular mechanism responsible for this regulation remains unclear. Here, we tested whether aldosterone represses NHE3 through microRNA-dependent posttranscriptional regulation. Transcriptomic analysis of distal colon from dietary Na+-depleted rats identified miR-204-5P (miR-204-5P) as markedly upregulated. Aldosterone increased miR-204-5P abundance and concomitantly reduced NHE3 mRNA, protein expression, and transport activity in rat and human distal colonic epithelium and in SK-CO15 cells. Bioinformatic and reporter analyses identified a conserved miR-204-5P binding site within the NHE3 3'-untranslated region, and miR-204-5P mimic transfection markedly suppressed NHE3 expression and transport activity without affecting other Na+/H+ exchanger isoforms. These findings identify a previously unrecognized aldosterone-microRNA signaling pathway that mediates chronic repression of NHE3 and provide new insight into hormonal regulation of colonic Na+ absorption.NEW & NOTEWORTHY This study identifies a previously unrecognized aldosterone-microRNA signaling mechanism regulating colonic Na+ absorption. We demonstrate that aldosterone induces miR-204-5P, which directly targets the NHE3 3'-untranslated region and suppresses NHE3 expression and transport activity in distal colonic epithelium. These findings reveal a microRNA-mediated pathway linking mineralocorticoid signaling to long-term inhibition of electroneutral NaCl absorption, providing new insight into hormonal regulation of intestinal electrolyte transport.

Animals↗

PubMed2004

Akt2, phosphatidylinositol 3-kinase, and PTEN are in lipid rafts of intestinal cells: role in absorption and differentiation.

BACKGROUND AND AIMS: In intestinal Na absorptive cells, phosphatidylinositol 3-kinase (PI 3-K) is involved in rapid epidermal growth factor (EGF) stimulation of Na absorption by the brush border membrane (BBM) Na(+)/H(+) exchanger NHE3. However, how NHE3 is regulated by the PI 3-K pathway and the role of Akt2 are poorly defined. METHODS: The localization of Akt, PI 3-K, and NHE3 was determined by either immunocytochemistry and/or membrane fractionation using OptiPrep density gradient centrifugation. RESULTS: In ileum, active total Akt was present most in the villi and basal layer of the crypts, and Akt2 was mostly in villi. In villus cells, PI 3-K and Akt2 were mostly at the apical surface at which they were present partially in lipid rafts (LR). EGF increased PI 3-K and active Akt2 in ileal BBM at the same time that it increased PI 3-K-dependent trafficking of NHE3 to BBM and stimulation of Na absorption. However, Akt2 was only active in the detergent soluble (DS) pool and not LR of ileal BBM, which correlated with the presence of PTEN in LR. In Caco-2 cells, while EGF stimulated BB NHE3, Akt2 was active in both LR and DS pools. This correlated with the lack of PTEN in the LR of Caco-2 membranes. Akt2 also correlated with epithelial cell differentiation. Akt2 amount and activity were greater in differentiated than undifferentiated Caco-2 cells. CONCLUSIONS: These results suggest that LR may play an important role in determining the function of PI 3-K/Akt2 signaling, including stimulation of intestinal Na absorption. These results also suggest that LR-associated Akt2 may be involved in enterocyte differentiation.

Animals↗

PubMed2003

Akt as a mediator of cell death.

Protein kinase B/Akt possesses prosurvival and antiapoptotic activities and is involved in growth factor-mediated neuronal protection. In this study we establish Akt deactivation as a causal mediator of cell death. Akt deactivation occurs in multiple models of cell death including N-methyl-d-aspartate excitotoxicity, vascular stroke, and nitric oxide (NO)- and hydrogen peroxide (H2O2)-elicited death of HeLa, PC12, and Jurkat T cells. Akt deactivation characterizes both caspase-dependent and -independent cell death. Conditions rescuing cell death, such as treatment with poly(ADP-ribose) polymerase or NO synthase inhibitors and preconditioning with sublethal concentrations of N-methyl-d-aspartate, restore Akt activity. Infection of neurons with adenovirus expressing constitutively active Akt prevents excitotoxicity, whereas phosphatidylinositol 3-kinase inhibitors or infection with dominant negative Akt induce death of untreated neuronal cells.

Animals↗

PubMed2003

Ca2+-dependent inhibition of NHE3 requires PKC alpha which binds to E3KARP to decrease surface NHE3 containing plasma membrane complexes.

The intestinal brush border (BB) Na+/H+ exchanger isoform 3 (NHE3) is acutely inhibited by elevation in the concentration of free intracellular Ca2+ ([Ca2+]i) by the cholinergic agonist carbachol and Ca2+ ionophores in a protein kinase C (PKC)-dependent manner. We previously showed that elevating [Ca2+]i with ionomycin rapidly inhibited NHE3 activity and decreased the amount of NHE3 on the plasma membrane in a manner that depended on the presence of the PDZ domain-containing protein E3KARP (NHE3 kinase A regulatory protein, also called NHERF2). The current studies were performed in PS120 fibroblasts (NHE-null cell line) stably transfected with NHE3 and E3KARP to probe the mechanism of PKC involvement in Ca2+ regulation of NHE3. Pretreatment with the general PKC inhibitor, GF109203X prevented ionomycin inhibition of NHE3 without altering basal NHE3 activity. Similarly, the Ca2+-mediated inhibition of NHE3 activity was blocked after pretreatment with the conventional PKC inhibitor Gö-6976 and a specific PKCalpha pseudosubstrate-derived inhibitor peptide. [Ca2+]i elevation caused translocation of PKCalpha from cytosol to membrane. PKCalpha bound to the PDZ1 domain of GST-E3KARP in vitro in a Ca2+-dependent manner. PKCalpha and E3KARP coimmunoprecipitated from cell lysates; this occurred to a lesser extent at basal [Ca2+]i and was increased with ionomycin exposure. Biotinylation studies demonstrated that [Ca2+]i elevation induced oligomerization of NHE3 in total lysates and decreased the amount of plasma membrane NHE3. Treatment with PKC inhibitors did not affect the oligomerization of NHE3 but did prevent the decrease in surface amount of NHE3. These results suggest that PKCalpha is not necessary for the Ca2+-dependent formation of the NHE3 plasma membrane complex, although it is necessary for decreasing the membrane amounts of NHE3, probably by stimulating NHE3 endocytosis.

Animals↗

PubMed2003

The protein kinase Akt induces epithelial mesenchymal transition and promotes enhanced motility and invasiveness of squamous cell carcinoma lines.

Epithelial-mesenchymal transition (EMT) is an important process during development and oncogenesis by which epithelial cells acquire fibroblast-like properties and show reduced intercellular adhesion and increased motility. Squamous cell carcinoma lines engineered to express constitutively active Akt underwent EMT, characterized by down-regulation of the epithelial markers desmoplakin, E-cadherin, and beta-catenin and up-regulation of the mesenchymal marker vimentin. The cells lost epithelial cell morphology and acquired fibroblast-like properties. Additionally, E-cadherin was down-regulated transcriptionally. The cells expressing constitutively active Akt exhibited reduced cell-cell adhesion, increased motility on fibronectin-coated surfaces, and increased invasiveness in animals. AKT is activated in many human carcinomas, and the AKT-driven EMT may confer the motility required for tissue invasion and metastasis. These findings suggest that future therapies based on AKT inhibition may complement conventional treatments by controlling tumor cell invasion and metastasis.

Cadherins↗

PubMed2003

Lysophosphatidic acid stimulates brush border Na+/H+ exchanger 3 (NHE3) activity by increasing its exocytosis by an NHE3 kinase A regulatory protein-dependent mechanism.

Na(+)/H(+) exchanger 3 (NHE3) kinase A regulatory protein (E3KARP) has been implicated in cAMP- and Ca(2+)-dependent inhibition of NHE3. In the current study, a new role of E3KARP is demonstrated in the stimulation of NHE3 activity. Lysophosphatidic acid (LPA) is a mediator of the restitution phase of inflammation but has not been studied for effects on sodium absorption. LPA has no effect on NHE3 activity in opossum kidney (OK) proximal tubule cells, which lack expression of endogenous E3KARP. However, in OK cells exogenously expressing E3KARP, LPA stimulated NHE3 activity. Consistent with the stimulatory effect on NHE3 activity, LPA treatment increased the surface NHE3 amount, which occurred by accelerating exocytic trafficking (endocytic recycling) to the apical plasma membrane. These LPA effects only occurred in OK cells transfected with E3KARP. The LPA-induced increases of NHE3 activity, surface NHE3 amounts, and exocytosis were completely inhibited by pretreatment with the PI 3-kinase inhibitor, LY294002. LPA stimulation of the phosphorylation of Akt was used as an assay for PI 3-kinase activity. LY294002 completely prevented the LPA-induced increase in Akt phosphorylation, which is consistent with the inhibitory effect of LY294002 on the LPA stimulation of NHE3 activity. The LPA-induced phosphorylation of Akt was the same in OK cells with and without E3KARP. These results show that LPA stimulates NHE3 in the apical surface of OK cells by a mechanism that is dependent on both E3KARP and PI 3-kinase. This is the first demonstration that rapid stimulation of NHE3 activity is dependent on an apical membrane PDZ domain protein.

Animals↗

PubMed2002

Na(+)/H(+ ) exchanger regulatory factor 2 directs parathyroid hormone 1 receptor signalling.

The parathyroid hormone 1 receptor (PTH1R) is a class II G-protein-coupled receptor. PTH1R agonists include both PTH, a hormone that regulates blood calcium and phosphate, and PTH-related protein (PTHrP), a paracrine/autocrine factor that is essential for development, particularly of the skeleton. Adenylyl cyclase activation is thought to be responsible for most cellular responses to PTH and PTHrP, although many actions appear to be independent of adenylyl cyclase. Here we show that the PTH1R binds to Na(+)/H(+) exchanger regulatory factors (NHERF) 1 and 2 through a PDZ-domain interaction in vitro and in PTH target cells. NHERF2 simultaneously binds phospholipase C beta 1 and an atypical, carboxyl-terminal PDZ consensus motif, ETVM, of the PTH1R through PDZ1 and PDZ2, respectively. PTH treatment of cells that express the NHERF2 PTH1R complex markedly activates phospholipase C beta and inhibits adenylyl cyclase through stimulation of inhibitory G proteins (G(i/o) proteins). NHERF-mediated assembly of PTH1R and phospholipase C beta is a unique mechanism to regulate PTH signalling in cells and membranes of polarized cells that express NHERF, which may account for many tissue- and cell-specific actions of PTH/PTHrP and may also be relevant to signalling by many G-protein-coupled receptors.

Adenylate Cyclase Toxin↗

PubMed2002

Ca(2+)-dependent inhibition of Na+/H+ exchanger 3 (NHE3) requires an NHE3-E3KARP-alpha-actinin-4 complex for oligomerization and endocytosis.

Two PDZ domain-containing proteins, NHERF and E3KARP are necessary for cAMP-dependent inhibition of Na(+)/H(+) exchanger 3 (NHE3). In this study, we demonstrate a specific role of E3KARP, which is not duplicated by NHERF, in Ca(2+)-dependent inhibition of NHE3 activity. NHE3 activity is inhibited by elevation of intracellular Ca(2+) ([Ca(2+)](i)) in PS120 fibroblasts stably expressing E3KARP but not those expressing NHERF. In addition, this Ca(2+)-dependent inhibition requires Ca(2+)-dependent association between alpha-actinin-4 and E3KARP. NHE3 is indirectly connected to alpha-actinin-4 in a protein complex through Ca(2+)-dependent interaction between alpha-actinin-4 and E3KARP, which occurs through the actin-binding domain plus spectrin repeat domain of alpha-actinin-4. Elevation of [Ca(2+)](i) results in oligomerization and endocytosis of NHE3 as well as in inhibition of NHE3 activity. Overexpression of alpha-actinin-4 potentiates the inhibitory effect of ionomycin on NHE3 activity by accelerating the oligomerization and endocytosis of NHE3. In contrast, overexpression of the actin-binding domain plus spectrin repeat domain acts as a dominant-negative mutant and prevents the inhibitory effect of ionomycin on NHE3 activity as well as the oligomerization and internalization of NHE3. From these results, we propose that elevated Ca(2+) inhibits NHE3 activity through oligomerization and endocytosis of NHE3, which occurs via formation of an NHE3-E3KARP-alpha-actinin-4 complex.

Actinin↗

PubMed2002

The burden of selected digestive diseases in the United States.

BACKGROUND & AIMS: Gastrointestinal (GI) and liver diseases inflict a heavy economic burden. Although the burden is considerable, current and accessible information on the prevalence, morbidity, and cost is sparse. This study was undertaken to estimate the economic burden of GI and liver disease in the United States for use by policy makers, health care providers, and the public. METHODS: Data were extracted from a number of publicly available and proprietary national databases to determine the prevalence, direct costs, and indirect costs for 17 selected GI and liver diseases. Indirect cost calculations were purposefully very conservative. These costs were compared with National Institutes of Health (NIH) research expenditures for selected GI and liver diseases. RESULTS: The most prevalent diseases were non-food-borne gastroenteritis (135 million cases/year), food-borne illness (76 million), gastroesophageal reflux disease (GERD; 19 million), and irritable bowel syndrome (IBS; 15 million). The disease with the highest annual direct costs in the United States was GERD ($9.3 billion), followed by gallbladder disease ($5.8 billion), colorectal cancer ($4.8 billion), and peptic ulcer disease ($3.1 billion). The estimated direct costs for these 17 diseases in 1998 dollars were $36.0 billion, with estimated indirect costs of $22.8 billion. The estimated direct costs for all digestive diseases were $85.5 billion. Total NIH research expenditures were $676 million in 2000. CONCLUSIONS: GI and liver diseases exact heavy economic and social costs in the United States. Understanding the prevalence and costs of these diseases is important to help set priorities to reduce the burden of illness.

Cost of Illness↗

PubMed2002

Human Na(+)/H(+) exchanger isoform 6 is found in recycling endosomes of cells, not in mitochondria.

Since the discovery of the first intracellular Na(+)/H(+) exchanger in yeast, Nhx1, multiple homologs have been cloned and characterized in plants. Together, studies in these organisms demonstrate that Nhx1 is located in the prevacuolar/vacuolar compartment of cells where it sequesters Na(+) into the vacuole, regulates intravesicular pH, and contributes to vacuolar biogenesis. In contrast, the human homolog of Nhx1, Na(+)/H(+) exchanger isoform 6 (NHE6), has been reported to localize to mitochondria when transiently expressed as a fusion with green fluorescent protein. This result warrants reevaluation because it conflicts with predictions from phylogenetic analyses. Here we demonstrate that when epitope-tagged NHE6 is transiently expressed in cultured mammalian cells, it does not colocalize with mitochondrial markers. It also does not colocalize with markers of the lysosome, late endosome, trans-Golgi network, or Golgi cisternae. Rather, NHE6 is distributed in recycling compartments and transiently appears on the plasma membrane. These results suggest that, like its homologs in yeast and plants, NHE6 is an endosomal Na(+)/H(+) exchanger that may regulate intravesicular pH and volume and contribute to lysosomal biogenesis.

Animals↗