Construction Of Targeting Vector For Conditional Knockout Of Murine Pkhd1 Gene And Studies On Its Function

Autosomal recessive polycystic kidney disease (ARPKD) is animportant renal and hepatic disease in neonates and infants, occurring 1 in 20000-40 000 live births. The main pathologic manifestations of the disease arethe fusiform dilation of renal collecting ducts and distal tubules as well asdysgenesis of the hepatic portal triad including hyperplastic biliary ducts andcongenital hepatic fibrosis. In infancy, the disease results in significantlyenlarged polycystic kidneys and pulmonary hypoplasia. Up to 30% of theaffected neonates die of secondary respiratory failure shortly after birth. Insubset that survived the perinatal period, the morbidity and mortality aremainly due to severe systemic hypertension, renal failure, and portalhypertension. Therefore, the patients with ARPKD are candidates for liver,kidney or combined liver and kidney transplantation.In 1994, the gene for ARPKD was mapped to human chromosome6p21-cen. Then independent groups isolated the gene, and severaldisease-causing mutations in the PKHD1 gene were identified. The geneencodes a novel large transmembrane protein with 4074 amino acids namedfibrocystin or polyductin. Studies have shown that its mouse gene homologue Pkhd1 is expressed in ductal structures during development of kidney, liver,lung, pancreas, and vessels.In adult kidney, PKHD1 is expressed in theepithelia of proximal convoluted tubules, ascending limbs of Henle's loopand collecting ducts. The protein is localized in the basal bodies and primarycilia of renal epithelia. To date, however there have been few studies on thefunction of the large complex protein. Its function remains unknown. Presentstudy aimed to construct the targeting vector for conditional knockout forpolycystic kidney and hepatic disease 1 gene (Pkhd1) and to investigatewhether the inhibition of PKHD1 expression by gene silencing could increaseEGF-induced cell proliferation and its possible mechanisms. The results wereas the following:1. Construction of targeting vector for conditional knockout ofmurine Pkhd1 gene. To construct the targeting vector for conditionalknockout for polycystic kidney and hepatic disease 1 gene (Pkhd1), a 9.3 kbPkhd1 genomic DNA fragment containing exon 6, obtained from murinegenomic DNA(mouse 129/SvJ) by PCR,were inserted into T vector(pCR2.1-TOPO) and used as the homologous arm. Two LoxP was insertedinto intron 5 and 6 for conditional knockout of exon 6 of Pkhd1. The correctstructure of the targeting vector was confirmed by restriction enzymedigestion and sequencing analysis. A targeting vector for conditionalknockout of murine Pkhd1 has been successfully constructed.2. Establishment and characterization of stable PKHD1-silencedHEK-293T cell lines. The shRNA2 vector with the least PKHD1 mRNAlevel was selected to establish the stable PKHD1-silenced cell lines. Inparallel, we used pGenesil-2 plasmid and HK-A shRNA vector to transfect HEK-293T cells to produce respective stable control cell lines. It was foundthat the PKHD1 mRNA level was markedly decreased in the PKHD1-silenced cell lines but not in the controls while the PKHD1 mRNA level ofthe controls were similar to that of wild-type cells.These results showed thatPKHD1 was down-regulated by the shRNA in the established stable cell linesand the gene silencing is specific.3. Hyperproliferative response of PKHD1-silenced cell lines tostimulation with EGF. A significant increase of proliferation rates inPKHD1-silenced cells was observed after the stimulation with EGF atconcentrations of 20ng/ml, and the proliferation rates of control groups weresimilar to those of wild-type cells. The EGF treatment increased the level ofERK1/2 phosphorylation in all cell groups with the highest effect inPKHD1-silenced cell lines. And PD98059, an inhibitor for ERK1/2 activation,showed inhibitory effect on EGF-induced cell proliferation in all the four cellgroups. Thus the results suggest that the hyperproliferative effect of EGF ismediated by ERK1/2 in PKHD1-silenced cells.4. PKHD1-silencing decreases the intracellular Ca²⁺ concentration inHEK 293T cells. In our experiments, the intracellular Ca²⁺ concentration ofthem was significantly lower than that of wild-type HEK-293T cells(P＜0.001), but no significant difference between control groups and wild-typecells was observed.5. Disturbed intracellular Ca²⁺ homeostasis due to knock-down ofPKHD1 caused EGF-induced cell overproliferation in thePKHD1-silenced cell though increasing ERK1/2 activation. Verapamiltreatment caused a stable decrease of intracellular calcium after 1 hour in HEK-293T cells and this was dose-dependent. When the cells were treatedwith verapamil for 4 hours, and then incubated in medium with 20ng EGF for24 hours, the cell proliferation rates were significantly increased. Also, theverapamil treatment increased the level of activated ERK1/2 in theEGF-treated cells. However, the increase of intracellular Ca²⁺ by Bay K8644represses the proliferative response to EGF. Meanwhile, ERK1/2overactivation was repressed by Bay K8644.In conclusion, the construction of targeting vector paved the way forconditional knockout mouse strain generated by targeted mutation of Pkhd1.these data also emphasize the role of Ca²⁺ regulation in pathogenesis ofARPKD and support that the fibrocystin may act as a membrane receptorparticipating in Ca²⁺ signaling. Thus, it may be postulated that the loweredintracellular Ca²⁺ leads to EGF-induced hyperproliferation in cells withsilenced PKHD1 and the loss of fibrocystin by the gene mutation causeshyperproliferation of kidney epithelial cells and cyst formation throughchange of intracellular Ca²⁺ concentration of kidney epithium in ARPKD.