Cholangiocyte Myosin IIB Is Required for Localized Aggregation of Sodium Glucose Cotransporter 1 to Sites of Cryptosporidium parvum Cellular Invasion and Facilitates Parasite Internalization ▿

Internalization of the obligate intracellular apicomplexan parasite, Cryptosporidium parvum, results in the formation of a unique intramembranous yet extracytoplasmic niche on the apical surfaces of host epithelial cells, a process that depends on host cell membrane extension. We previously demonstrated that efficient C. parvum invasion of biliary epithelial cells (cholangiocytes) requires host cell actin polymerization and localized membrane translocation/insertion of Na+/glucose cotransporter 1 (SGLT1) and of aquaporin 1 (Aqp1), a water channel, at the attachment site. The resultant localized water influx facilitates parasite cellular invasion by promoting host-cell membrane protrusion. However, the molecular mechanisms by which C. parvum induces membrane translocation/insertion of SGLT1/Aqp1 are obscure. We report here that cultured human cholangiocytes express several nonmuscle myosins, including myosins IIA and IIB. Moreover, C. parvum infection of cultured cholangiocytes results in the localized selective aggregation of myosin IIB but not myosin IIA at the region of parasite attachment, as assessed by dual-label immunofluorescence confocal microscopy. Concordantly, treatment of cells with the myosin light chain kinase inhibitor ML-7 or the myosin II-specific inhibitor blebbistatin or selective RNA-mediated repression of myosin IIB significantly inhibits (P < 0.05) C. parvum cellular invasion (by 60 to 80%). Furthermore ML-7 and blebbistatin significantly decrease (P < 0.02) C. parvum-induced accumulation of SGLT1 at infection sites (by approximately 80%). Thus, localized actomyosin-dependent membrane translocation of transporters/channels initiated by C. parvum is essential for membrane extension and parasite internalization, a phenomenon that may also be relevant to the mechanisms of cell membrane protrusion in general

Inhibition of Cdc25A Suppresses Hepato-Renal Cystogenesis in Rodent Models of Polycystic Kidney and Liver Disease

BACKGROUND & AIMS: In polycystic kidney (PKD) and liver (PLD) diseases, the normally non-proliferative hepato-renal epithelia acquire a proliferative, cystic phenotype, which is linked to overexpression of Cdc25A and cell cycle deregulation. We investigated the effects of Cdc25A inhibition in mice and rats, via genetic and pharmacological approaches. METHODS: Cdc25A(+/-) mice (which have reduced levels of Cdc25A) were cross-bred with Pkhd1(del2/del2) mice (which have increased levels of Cdc25A and develop hepatic cysts). Cdc25A expression was analyzed in livers of control and PCK rats, control and Pkd2(ws25/-) mice, healthy individuals, and patients with PLD. We examined effects of pharmacologic inhibition of Cdc25A with Vitamin K3 (VK3) on the cell cycle, proliferation, and cyst expansion in vitro; hepato-renal cystogenesis in PCK rats and Pkd2(ws25/-) mice; and expression of Cdc25A and the cell cycle proteins regulated by Cdc25A. We also examined effects of the Cdc25A inhibitor PM-20 on hepato-renal cystogenesis in Pkd2(ws25/-) mice. RESULTS: Liver weights and hepatic and fibrotic areas were decreased by 32%-52% in Cdc25A(+/-):Pkhd1(del2/del2) mice, compared to Pkhd1(del2/del2) mice.VK3 altered the cell cycle and reduced proliferation of cultured cholangiocytes by 32%-83% and decreased growth of cultured cysts by 23%-67%. In PCK rats and Pkd2(ws25/-) mice, VK3 reduced liver and kidney weights and hepato-renal cystic and fibrotic areas by 18%-34%. PM-20 decreased hepato-renal cystogenesis in Pkd2(ws25/-) mice by 15%. CONCLUSIONS: Cdc25A inhibitors block cell cycle progression and proliferation, reduce liver and kidney weights and cyst growth in animal models of PKD and PLD, and might be developed as therapeutics for these diseases