Suhasini Ganta

Altered ion transport by thyroid epithelia from CFTR−/− pigs suggests mechanisms for hypothyroidism in cystic fibrosis

Subclinical hypothyroidism has been linked to cystic fibrosis, and the cystic fibrosis transmembrane conductance regulator (CFTR) shown to be expressed in the thyroid. The thyroid epithelium secretes Cl− and absorbs Na+ in response to cAMP. Chloride secretion may provide a counter‐ion for the SLC26A4 (pendrin)‐mediated I− secretion which is required for the first step of thyroid hormonogenesis, thyroglobulin iodination. In contrast, few models exist to explain a role for Na+ absorption. Whether CFTR mediates the secretory Cl− current in thyroid epithelium has not been directly addressed. We used thyroids from a novel pig CFTR−/− model, generated primary pig thyroid epithelial cell cultures (pThECs), analysed these cultures for preservation of thyroid‐specific transcripts and proteins, and monitored the following parameters: (1) the Cl− secretory response to the cAMP agonist, isoprenaline; and (2) the amiloride‐sensitive Na+ current. Baseline short‐circuit current (Isc) did not differ between CFTR+/+ and CFTR−/− cultures. Serosal isoprenaline increased Isc in CFTR+/+, but not CFTR−/−, monolayers. Compared with CFTR+/+ thyroid cultures, amiloride‐sensitive Na+ absorption measured in CFTR−/− pThECs represented a greater fraction of the resting Isc. However, levels of transcripts encoding epithelial sodium channel (ENaC) subunits did not differ between CFTR+/+ and CFTR−/− pThECs. Immunoblot analysis verified ENaC subunit protein expression, but quantification indicated no difference in expression levels. Our studies definitively demonstrate that CFTR mediates cAMP‐stimulated Cl− secretion in a well‐differentiated thyroid culture model and that knockout of CFTR promotes increased Na+ absorption by a mechanism other than increased ENaC expression. These findings suggest several models for the mechanism of cystic fibrosis‐associated hypothyroidism.

FSH stimulates ovarian cancer cell growth by action on growth factor variant receptor.

A number of FSH receptor (FSH-R) isoforms with distinct structural motifs and signaling paradigms have been described, including a single transmembrane domain variant that functions as a growth factor type receptor (FSH-R3). This study tested the hypothesis that FSH can stimulate ovarian cancer cell proliferation by acting on FSH-R3, using the tumorigenic mouse ovarian surface epithelial cell (MOSEC) line ID8. FSH enhanced ID8 proliferation in a concentration-dependent fashion. Moreover, FSH-treatment of ID8 elicited intracellular events consistent with activation of FSH-R3 and distinct from those associated with activation of the canonical G-protein coupled FSH-R isoform (FSH-R1). Specifically, the FSH-R3 signaling pathway included cAMP-independent activation of ERK downstream of an SNX-482 sensitive component likely to be the Cav2.3 calcium channel. Northern analysis using probes specific for exons 7 and 11 of FSH-R identified consistently only one 1.9kb transcript. Immunoblot analysis confirmed expression of FSH-R3 but not FSHR-1 in ID8. Together, these data suggest that FSH-R3 signaling promotes proliferation of ovarian cancer cells.

Drug-Induced Alterations to Gene and Protein Expression in Intestinal Epithelial Cell 6 Cells Suggest a Role for Calpains in the Gastrointestinal Toxicity of Nonsteroidal Anti-Inflammatory Agents

Nonsteroidal anti-inflammatory drugs (NSAIDs) are used extensively as therapeutic agents, despite their well documented gastrointestinal (GI) toxicity. At this time, the mechanisms responsible for NSAID-associated GI damage are incompletely understood. In this study, we used microarray analysis to generate a novel hypothesis about cellular mechanisms that underlie the GI toxicity of NSAIDs. Monolayers of intestinal epithelial cells (IEC-6) were treated with NSAIDs that either exhibit (indomethacin, NS-398 [N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide]) or lack (SC-560 [5-(4-chlorphenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole]) inhibitory effects on IEC-6 migration. Bioinformatic analysis of array data identified the calpain cysteine proteases and their endogenous inhibitor calpastatin as potential targets of NSAIDs shown previously to retard IEC-6 migration. Accordingly, quantitative real-time reverse transcription polymerase chain reaction and immunoblotting were performed to assess the effects of NSAIDs on the expression of mRNA and protein for calpain 8, calpain 2, calpain 1, and calpastatin. In treated IEC-6 monolayers, NS-398 decreased the expression of mRNA for calpain 2 and calpain 8. Both NS-398 and indomethacin decreased the protein expression of calpains 8, 2, and 1. None of the NSAIDs affected expression of calpastatin mRNA or protein. The calpain inhibitors, N-acetyl-Leu-Leu-methioninal and N-acetyl-Leu-Leu-Nle-CHO, retarded IEC-6 cell migration in a concentration-dependant fashion, and these inhibitory effects were additive with those of indomethacin and NS-398. Our experimental results suggest that the altered expression of calpain proteins may contribute to the adverse effects of NSAIDs on intestinal epithelial restitution.

4-Aminopyridine Decreases Progesterone Production by Porcine Granulosa Cells

BackgroundIon channels occur as large families of related genes with cell-specific expression patterns. Granulosa cells have been shown to express voltage-gated potassium channels from more than one family. The purpose of this study was to determine the effects of 4-aminopyridine (4-AP), an antagonist of KCNA but not KCNQ channels.MethodsGranulosa cells were isolated from pig follicles and cultured with 4-AP, alone or in combination with FSH, 8-CPT-cAMP, estradiol 17β, and DIDS. Complimentary experiments determined the effects of 4-AP on the spontaneously established pig granulosa cell line PGC-2. Granulosa cell or PGC-2 function was assessed by radio-immunoassay of media progesterone accumulation. Cell viability was assessed by trypan blue exclusion. Drug-induced changes in cell membrane potential and intracellular potassium concentration were documented by spectrophotometric determination of DiBAC4(3) and PBFI fluorescence, respectively. Expression of proliferating cell nuclear antigen (PCNA) and steroidogenic acute regulatory protein (StAR) was assessed by immunoblotting. Flow cytometry was also used to examine granulosa cell viability and size.Results4-AP (2 mM) decreased progesterone accumulation in the media of serum-supplemented and serum-free granulosa cultures, but inhibited cell proliferation only under serum-free conditions. 4-AP decreased the expression of StAR, the production of cAMP and the synthesis of estradiol by PGC-2. Addition of either 8-CPT-cAMP or estradiol 17β to serum-supplemented primary cultures reduced the inhibitory effects of 4-AP. 4-AP treatment was also associated with increased cell size, increased intracellular potassium concentration, and hyperpolarization of resting membrane potential. The drug-induced hyperpolarization of resting membrane potential was prevented either by decreasing extracellular chloride or by adding DIDS to the media. DIDS also prevented 4-AP inhibition of progesterone production.Conclusion4-AP inhibits basal and FSH-stimulated progesterone production by pig granulosa cells via drug action at multiple interacting steps in the steroidogenic pathway. These inhibitory effects of 4-AP on steroidogenesis may reflect drug-induced changes in intracellular concentrations of K+and Cl- as well as granulosa cell resting membrane potential.

Attenuated Mutants of Ehrlichia chaffeensis Induce Protection against Wild-Type Infection Challenge in the Reservoir Host and in an Incidental Host

ABSTRACT Ehrlichia chaffeensis, a tick-borne rickettsial organism, causes the disease human monocytic ehrlichiosis. The pathogen also causes disease in several other vertebrates, including dogs and deer. In this study, we assessed two clonally purified E. chaffeensis mutants with insertions within the genes Ech_0379 and Ech_0660 as vaccine candidates in deer and dogs. Infection with the Ech_0379 mutant and challenge with wild-type E. chaffeensis 1 month following inoculation with the mutant resulted in the reduced presence of the organism in blood compared to the presence of wild-type infection in both deer and dogs. The Ech_0660 mutant infection resulted in its rapid clearance from the bloodstream. The wild-type infection challenge following Ech_0660 mutant inoculation also caused the pathogens clearance from blood and tissue samples as assessed at the end of the study. The Ech_0379 mutant-infected and -challenged animals also remained positive for the organism in tissue samples in deer but not in dogs. This is the first study that documents that insertion mutations in E. chaffeensis that cause attenuated growth confer protection against wild-type infection challenge. This study is important in developing vaccines to protect animals and people against Ehrlichia species infections.

Endogenous surface expression of ΔF508‐CFTR mediates cAMP‐stimulated Cl− current in CFTRΔF508/ΔF508 pig thyroid epithelial cells

The cystic fibrosis transmembrane conductance regulator (CFTR) is both an anion channel and a regulator of other transport proteins. Mutations in the CFTR gene underlie the human disease, cystic fibrosis. The most common CFTR mutation, ΔF508, produces a misfolded protein which traffics improperly. The availability of transgenic CFTRΔF508/ΔF508 pigs allows measurement of the impact of ΔF508 in native tissue. Thyroid epithelia respond to cAMP‐elevating agents by increasing anion transport, a process reliant on functional CFTR. To assess whether endogenous levels of ΔF508‐CFTR mediate thyroid transport, primary thyroid epithelial cultures (pThECs) were grown from newborn CFTR+/+ (wild‐type) and CFTRΔF508/ΔF508 (ΔF) pig thyroids and the stimulated, secretory components of short‐circuit current (Isc) compared. Surface biotinylation studies assessed the surface presentation of ΔF508‐CFTR. Baseline Isc levels of both wild‐type and ΔF pThECs consisted of an amiloride‐sensitive component. In ΔF pThECs, this mirrored previous measurements in CFTR−/− (knockout) pThECs. Surprisingly, elevation of cAMP transiently increased Isc to peak levels ∼65% of those achieved by wild‐type. In contrast, knockout pThECs were indifferent to cAMP activation. In ΔF pThECs, total ΔF508‐CFTR expression was ∼9% that of wild‐type, consistent with misfolding and enhanced degradation. Surface biotinylation studies indicated that ∼4% of the total ΔF508 resided at the surface and did not increase with cAMP elevation. The present findings show that low endogenous levels of pig ΔF508‐CFTR can mediate substantial anion transport by thyroid epithelia. These data suggest that both wild‐type and ΔF508‐CFTR regulate additional thyroid transporters, and together co‐ordinate the overall Isc response.