Conditional mutation of Pkd2 causes cystogenesis and upregulates beta-catenin

Abstract

Loss of polycystin-2 (PC2) in mice (Pkd2(-/-)) results in total body edema, focal hemorrhage, structural cardiac defects, abnormal left-right axis, hepatorenal and pancreatic cysts, and embryonic lethality. The molecular mechanisms by which loss of PC2 leads to these phenotypes remain unknown. We generated a model to allow targeted Pkd2 inactivation using the Cre-loxP system. Global inactivation of Pkd2 produced a phenotype identical to Pkd2(-/-) mice with undetectable PC2 protein and perinatal lethality. Using various Cre mouse lines, we found that kidney, pancreas, or time-specific deletion of Pkd2 led to cyst formation. In addition, we developed an immortalized renal collecting duct cell line with inactive Pkd2; these cells had aberrant cell-cell contact, ciliogenesis, and tubulomorphogenesis. They also significantly upregulated beta-catenin, axin2, and cMyc. Our results suggest that loss of PC2 disrupts normal behavior of renal epithelial cells through dysregulation of beta-catenin-dependent signaling, revealing a potential role for this signaling pathway in PC2-associated ADPKD.

Figure 1.

Pkd2 conditional knockout construct and molecular analysis of the specific targeting event at the Pkd2 locus. (A) Schematic representation of Cre-mediated gene-targeting strategy. (I) Pkd2^nf3 allele: There is a pGKNeo cassette flanked by two FRT sites at intron 2 of Pkd2, whereas Pkd2-exon-3 is flanked by two loxP sites. (II) Pkd2^f3 allele: The pGKNeo cassette at the Pkd2^nf3 allele was excised under mediation of Flpe recombinase and converted to the Pkd2^f3 allele, in which there are only two loxP sites flanking Pkd2-exon-3. (III) Pkd2^d3 allele: Pkd2-exon-3 is excised by mediation of Cre recombinase to produce Pkd2^d3 allele. X, XbaI; B, BglII. (B) Tail biopsy DNAs from mice with Pkd2^f3 allele(s) were digested with BglII and hybridized with probe B from outside the targeted region (Figure 1AI). The expected 15-kb wild-type band was observed in wild-type and Pkd2^f3 heterozygous mice. A mutant 12-kb band was seen in Pkd2^f3 heterozygous and homozygous mice. (C) Quantitative PCR to test Pkd2 mRNA expression level of E13.5 embryos with Pkd2^−/− (Pkd2^tm2som), Pkd2^d3/d3, Pkd2^+/f3, and Pkd2^f3/f3 alleles compared with a wild-type embryo. No significant change was observed between Pkd2^f3/f3 and wild-type alleles. Virtually no Pkd2 mRNA was seen in Pkd2^−/− or Pkd2^d3/d3 embryos. (D) A monoclonal anti-PC2 antibody, hPKD2-Cm1A11, which recognizes the intracellular COOH-terminal portion of PC2, was used to detect PC2 in E13.5 embryos with wild-type, Pkd2^+/f3, Pkd2^f3/f3, and Pkd2^d3/d3 alleles by Western blots. PC2 is absent in embryos with Pkd2^d3/d3 alleles, whereas there is no significant PC2 expression change in either Pkd2^+/f3 or Pkd2^f3/f3 compared with wild-type littermates. (E) PCR genotyping mice with Pkd2^d3 heterozygous and homozygous alleles. A 540-bp PCR band was observed for the Pkd2^d3 allele whereas a 350-bp PCR band was observed for the wild type.

Figure 2.

Temporal inactivation of the floxed Pkd2 allele induces cyst formation in the kidneys, liver, and pancreas. (A) A gross cystic kidney (arrow) is seen in a 9-wk-old γGT-Cre::Pkd2^f3/− mouse. (B) A wild-type gross kidney from its littermate acts as a normal control. (C) A 6-wk-old Mx1-Cre::Pkd2^flox3/− mouse received intraperitoneal injections of 500 μg pI-pC for 3 consecutive days and was dissected and examined after 8 wk. Cysts (arrow) are seen in the kidney of the Mx1-Cre::Pkd2^flox3/− mouse. (D) A wild-type gross kidney from its littermate acts as a normal control. (E) A 6-wk-old Pdx1-Cre::Pkd2^f3/− mouse received intraperitoneal injections of 1 mg Tamoxifen for 5 consecutive days and the pancreas was removed and examined 4 wk after injection. Multiple gross cysts (arrow) in the mouse pancreas were observed. Kidney sections, which correspond to (A through C), are shown in (F through H), respectively. (I) The same mouse shown in C also presented a great number of cysts in the liver (arrow). (J) There are multiple pancreatic cysts in the histologic section (arrow). Bars represent 0.2 mm in A and B and F through H; 0.5 mm in C through E; 5 μm in I; and 20 μm in J.

Figure 3.

Isolation of Pkd2^f3/− renal collecting duct cell lines and generation of the cell lines with null-Pkd2 alleles (Pkd2^d3/−). (A) Pkd2^f3/− cell line D3 was characterized by staining with the epithelia cell markers (a) cytokeratin and (b) E-cadherin and a collecting duct cell marker (c) DBA. Positive staining is seen in cultured D3 cell line for all three makers. (B) After AdCre infection, several single clones were isolated from the AdCre-infected D3 cell pool. The D3 daughter cell lines E8, C1, B2, and B3 were genotyped by PCR, in which a pair of primers is anchored outside regions of the loxP-exon3-loxP cassette (Figure 1AII). A 550-bp PCR band can be detected in cell lines with the Pkd2^d3 allele (i.e., E8, C1, and B2 in upper panel). No PCR product of this size was detected in some cell lines (such as B3), indicating the probability that a Pkd2^f3 allele is present. Because the D3 cell line carries a Pkd2⁻ (Pkd2^tm2som) allele, its daughter cell lines (i.e., B3, B2, C1, and E8) should contain Pkd2^+/− alleles. PCR genotyping showed that all daughter cell lines tested contained the wild-type 350-bp product and mutant 170-bp product (lower panel in B). (C) Western blot analyses confirmed the genotypes of these cell lines. Cell lines with Pkd2^f3/− alleles (such as mother cell line D3 and its daughter cell line B3 with its remaining Pkd2^f3 allele) showed positive immunoactivity for PC2 (approximately 110 kD), whereas no PC2 band was detected in cell lines with Pkd2^d3/− alleles (i.e., E8, C1, and B2). Bar represents 5 μm in A.

Figure 4.

Loss of PC2 impairs tubulomorphogenesis and disrupts normal cell-cell contact in vitro. (A) Phase contrast photomicrographs were taken from examples of Pkd2^f3/− D3 cells and Pkd2^d3/− E8 cells cultured for 7 d in 3D Matrigel gels. Tubulomorphogenesis was seen in 3D cultured Pkd2^f3/− D3 cells (left), but not in its daughter Pkd2^d3/- E8 cells (right). (B) Quantification of the tubulomorphogenic results from 3D cultured Pkd2^f3/− D3 and Pkd2^d3/− E8 cells. Approximately 35% of Pkd2^f3/− D3 cells generate five or more tubular branches and less than 2% exhibit cell aggregation. Under similar conditions, 75% of Pkd2^d3/− E8 cells display cell aggregation and none formed five or more tubular branches. (C) IF staining of ZO-1 (green) showed a nearly normal view of tight junctions in cultured Pkd2^f3/− D3 cells (left), whereas this normal tight junction is disrupted in Pkd2^d3/− E8 cells (right). A ciliary marker anti-acetylated α-tubulin antibody (red) was used for IF co-staining. (D) Cultured Pkd2^f3/− D3 and Pkd2^d3/− E8 cells were stained with an anti-E-cadherin antibody (red). Compared with D3 cells (left), a diffuse E-cadherin distribution was seen in cultured E8 cells (right). DAPI dye (blue) was utilized for nucleic acid staining. Bar represents 20 μm in A and 5 μm in C through D.

Figure 5.

Loss of PC2 induces aberrant ciliogenesis in null-Pkd2 renal cells and tissues. (A) A common ciliary marker anti-acetylated α-tubulin antibody was used for IF staining of the Pkd2^f3/- D3 cell line. Confocal images show ciliary structures (arrows) in top (upper in A) and lateral views (lower in A). (B) Simultaneous IF staining of the Pkd2^d3/- cell line E8, results in ciliary structures that were shorter in size and fewer in number than Pkd2^f3/- D3 cells (arrows). (C) One hundred individual cells in five random high-power fields (1000×) from cell lines D3 and E8 were numbered, and the numbered cells were rated for the presence or absence of cilium-staining. In Pkd2^f3/− D3 cells, 40% of cells stained positive for cilia, compared with 20% of Pkd2^d3/− E8 cells (*P < 0.05). (D) The length of 50 individual primary cilia of these cultured cells from three random high-power fields was measured using lateral views of the confocal images; the average length of the primary cilia was calculated. The primary cilium length is approximately 3 μm in Pkd2^f3/− D3 cells and approximately 1 μm in Pkd2^f3/− D3 cells (*P < 0.05). (E) An anti-acetylated α-tubulin antibody was used to stain kidney sections from 12-mo-old Pkd2^nf3/nf3 mice and its wild-type littermates. Ciliary structures (arrows) were abundantly observed in wild-type kidneys (a and c). Cilia were fewer and shorter (arrows) in the corresponding cortical region of Pkd2^nf3/nf3 kidneys (a versus b). Similar reductions in number and length of ciliary structures (arrows) were observed in the corresponding medullary region of Pkd2^nf3/nf3 kidneys (c versus d). Bars represent 5 μm in A, B, Ea, and Eb; and 10 μm in Ec and Ed.

Figure 6.

Characteristics of proliferation and apoptosis in the null-Pkd2 renal epithelial cell lines. (A) Pkd2^f3/− cell lines D3 and B3 and Pkd2^d3/− B2 and E8 were incubated with ³H-thymidine after which the rate of ³H-thymidine incorporation was determined as described in the Concise Methods section. Null-Pkd2 cells (B2 and E8) showed significantly higher ³H-thymidine values than Pkd2^f3/− cells (D3 and B3) (*P < 0.05), a result analogous to previous observations that loss of PC2 promotes renal epithelial proliferation. (B) A TUNEL assay was also used to assess apoptosis. Null-Pkd2 cells (B2 and E8) showed a significantly higher percentage of cells undergoing apoptosis than Pkd2^f3/− cells (D3 and B3) (*P < 0.05).

Figure 7.

Loss of PC2 induces cyst formation by dysregulating β-catenin-dependent signaling. (A) Western analyses showed that β-catenin and axin2 were significantly upregulated in null-Pkd2 cells (B2 and E8), as compared with their mother Pkd2^f3/− cell line D3. An anti-PC2 antibody, hPKD2-Cm1A11, was used to evaluate the level of PC2 expression in the various cell lines. (B) A normalized quantitative analysis of the densitometry values of β-catenin and axin2 observed from B2/E8 or D3/B3 cell lines using results from at least three Western blots. The statistical analysis indicates that loss of PC2 significantly upregulates β-catenin expression levels (*P < 0.05). There is also a significant increase in axin2 expression in null-Pkd2 B2/E8 cell lines compared with Pkd2^f3/− D3/B3 cell lines (*P < 0.05). (C) Wnt3a was added to culture medium for 0, 24 (1 d), or 48 (2 d) h. Equal amounts of cell lysates were separated by SDS-PAGE and transferred to nitrocellulose membranes. Western blots analysis using antibodies directed toward axin2, β-catenin, and cMyc indicate that an inducible upregulated pattern is observed for axin2, β-catenin, and cMyc proteins in the Pkd2^f3/− D3 cell line. In contrast, the Pkd2^d3/− B2 cell line has a high constitutive level of axin2, β-catenin, and cMyc that is not altered with application of Wnt3a. (D) A panel of Western blots was performed for detection of cytosolic β-catenin (upper panel) and axin2 (lower panel). Compared with the Pkd2^f3/− D3 cell line, both Pkd2^d3/− cell lines (B2 and E8) showed an increased expression for β-catenin and axin2. (E) A normalized quantitative analysis for cytosolic β-catenin and axin2 expression levels in Pkd2^f3/− D3 and Pkd2^d3/− B2/E8 cell lines indicates that loss of PC2 significantly upregulates cytosolic β-catenin and axin2 (*P < 0.05). (F) IHC staining of β-catenin, axin2, and cMyc expression in the kidney of E13.5 embryonic littermates. A β-catenin antibody was used to stain the kidneys of (a) wild type and (b) Pkd2^d3/d3. Positive staining can be seen in renal epithelia of the Pkd2^d3/d3 kidney (arrows). A similar observation was observed with axin2 (c versus d) and cMyc (e versus f) antibody staining. (G) IHC staining of β-catenin, axin2, and cMyc expression in the adult kidney of Mx1-Cre::Pkd2^f3/− and Pkd2^f3/− littermates. Compared with Pkd2^f3/− littermates, more positive staining can be seen in renal epithelia of the Mx1-Cre::Pkd2^f3/− kidney (arrows) (a versus b). Similar observations were also noted for axin2 (c versus d) and cMyc (e versus f) antibody staining (arrows). The α-tubulin or β-actin Western blots in A, C, and D were used as a protein loading control. Bars represent 10 μm in Fa through Fd and 5 μm in Fe, Ff, and G.