Sarker R.,Gastroenterology and Hepatology Division |
Valkhoff V.E.,Gastroenterology and Hepatology Division |
Valkhoff V.E.,Erasmus Medical Center |
Zachos N.C.,Gastroenterology and Hepatology Division |
And 10 more authors.
American Journal of Physiology - Cell Physiology | Year: 2011
Na +/H + exchanger 3 (NHE3) is expressed in the brush border (BB) of intestinal epithelial cells and accounts for the majority of neutral NaCl absorption. It has been shown that the Na +/H + exchanger regulatory factor (NHERF) family members of multi-PDZ domain-containing scaffold proteins bind to the NHE3 COOH terminus and play necessary roles in NHE3 regulation in intestinal epithelial cells. Most studies of NHE3 regulation have been in cell models in which NHERF1 and/or NHERF2 were overexpressed. We have now developed an intestinal Na + absorptive cell model in Caco-2/bbe cells by expressing hemagglutinin (HA)-tagged NHE3 with an adenoviral infection system. Roles of NHERF1 and NHERF2 in NHE3 regulation were determined, including inhibition by cAMP, cGMP, and Ca 2+ and stimulation by EGF, with knockdown (KD) approaches with lentivirus (Lenti)-short hairpin RNA (shRNA) and/or adenovirus (Adeno)-small interfering RNA (siRNA). Stable infection of Caco-2/bbe cells by NHERF1 or NHERF2 Lenti-shRNA significantly and specifically reduced NHERF protein expression by >80%. NHERF1 KD reduced basal NHE3 activity, while NHERF2 KD stimulated NHE3 activity. siRNA-mediated (transient) and Lenti-shRNA-mediated (stable) gene silencing of NHERF2 (but not of NHERF1) abolished cGMP- and Ca 2+-dependent inhibition of NHE3. KD of NHERF1 or NHERF2 alone had no effect on cAMP inhibition of NHE3, but KD of both simultaneously abolished the effect of cAMP. The stimulatory effect of EGF on NHE3 was eliminated in NHERF1-KD but occurred normally in NHERF2-KD cells. These findings show that both NHERF2 and NHERF1 are involved in setting NHE3 activity. NHERF2 is necessary for cGMP-dependent protein kinase (cGK) II- and Ca 2+-dependent inhibition of NHE3. cAMP-dependent inhibition of NHE3 activity requires either NHERF1 or NHERF2. Stimulation of NHE3 activity by EGF is NHERF1 dependent. © 2011 the American Physiological Society.
Smedile A.,Gastroenterology and Hepatology Division |
Rizzetto M.,Gastroenterology and Hepatology Division
Digestive and Liver Disease | Year: 2011
The hepatitis D virus (HDV) was discovered in Italy in the mid-1970s during a major outbreak of hepatitis D in the Mediterranean basin. The outbreak has been brought under control in Europe and throughout the industrialized world in the last twenty years; in parallel, the clinical pattern of HDV disease has consistently changed.Though the decline of hepatitis D has diminished attention to this problem and at present testing for HDV is not seldom neglected, hepatitis D is not eradicated in Europe and its circulation did not decline further in the last decade. Fresh new cases are cumulating in migrants from HDV endemic areas of the developing world.Hepatitis D remains a major health problem in many developing areas with outbreaks of the disease continuing to be reported from Asia, Africa and South Africa. © 2011 Editrice Gastroenterologica Italiana S.r.l.