Characterization of gap junction proteins in the bladder of Cx43 mutant mouse models of oculodentodigital dysplasia

Abstract

Oculodentodigital dysplasia (ODDD) is a rare developmental disease resulting from germline mutations in the GJA1 gene that encodes the gap junction protein connexin43 (Cx43). In addition to the classical ODDD symptoms that affect the eyes, teeth, bone and digits, in some cases ODDD patients have reported bladder impairments. Thus, we chose to characterize the bladder in mutant mouse models of ODDD that harbor two distinct Cx43 mutations, G60S and I130T. Histological assessment revealed no difference in bladder detrusor wall thickness in mutant compared to littermate control mice. The overall localization of Cx43 in the lamina propria and detrusor also appeared to be similar in the bladders of mutant mice with the exception that the G60S mice had more instances of intracellular Cx43. However, both mutant mouse lines exhibited a significant reduction in the phosphorylated P1 and P2 isoforms of Cx43, while only the I130T mice exhibited a reduction in total Cx43 levels. Interestingly, Cx26 levels and distribution were not altered in mutant mice as it was localized to intracellular compartments and restricted to the basal cell layers of the urothelium. Our studies suggest that these two distinct genetically modified mouse models of ODDD probably mimic patients who lack bladder defects or other factors, such as aging or co-morbidities, are necessary to reveal a bladder phenotype.

Fig. 1

G60S and I130T mouse bladders have similar histology and c detrusor thickness as their wild-type littermate controls. Paraffin-embedded bladders were sectioned, stained with H and E and measured for detrusor thickness. In all mice, bladder histology was similar between mutant and matched wild-type (Wt) littermate controls (a, b). Subsequent detrusor measurements revealed that G60S and I130T mouse bladders have similar detrusor thicknesses as their respective wild-type littermates (c). Bars 200 μm (a, b), error bars ± SEM (c). n = 4 mice

Fig. 2

G60S and I130T mouse bladders exhibit a reduction in the c phosphorylated species of Cx43. Cx43 normally resolves as a triplet band representing different phosphorylated species of Cx43 (P0, P1 and P2), as seen in heart tissue (a). Cx43 was detected in the bladders of G60S and I130T mutant mice, as well as in wild-type (Wt) littermate mouse bladders but not in the mouse liver (a). Total Cx43 (P0, P1 and P2) expression was lower in I130T mouse bladders compared to littermate controls (b), a condition not observed in G60S mouse bladders (c). Lower levels of the phosphorylated Cx43 species (P1 and P2) were found in I130T (d) and G60S (e) mouse bladders compared to wild-type littermates. Nonspecific bands were noted at ~47–50 and ~30 kDa. Bars represent ±SEM (b–e). *P < 0.05. n = 5 (I130T mice) and 6 (G60S mice). AU arbitrary units

Fig. 3

The distribution of Cx43 in the lamina propria of wild-type and mutant mice. Cx43 was localized in the lamina propria of G60S (a) and I130T (b) mutant mice and compared to wild-type (Wt) littermate mouse bladder controls. G60S mouse bladders displayed significantly more intracellular Cx43 (arrows), as opposed to the punctate distribution (arrowheads) seen in wild-type littermates (a, histogram). I130T mouse bladders displayed a similar punctate Cx43 distribution pattern (arrowheads) as seen in wild-type littermates (b). Uro urothelium, LP lamina propria. Red indicates Cx43, blue indicates Hoechst nuclear stain. Bars 50 μm. Histogram error bars represent ±SEM. **P <0.01

Fig. 4

Cx43 was localized to resident cell types of the detrusor in G60S and I130T mutant mice and their wild-type littermates. In the detrusor layer, immunolabeling revealed that Cx43 was predominantly localized to cells of connective tissue surrounding smooth muscle bundles and smooth muscle cells in G60S (a) and I130T (b) mice and their respective wild-type (Wt) littermates. Cx43 displays punctate structures (arrowheads) in G60S (a), I130T (b) and wild-type littermates. Red indicates Cx43, green indicates F-actin, and blue indicates Hoechst nuclear stain. Bar 50 μm

Fig. 5

G60S and I130T mutant mouse bladders have similar Cx26 levels as wild-type littermates. Cx26 generally resolves at ~21 kDa, with the dimer resolving at ~37 kDa as seen in liver lysates (a). G60S, I130T and wild-type (Wt) littermate mouse bladders were all positive for Cx26 expression (a). Total Cx26 expression was similar in I130T and G60S mice and their wild-type littermates (b, c). A nonspecific band located at ~32–34 kDa was noted. Bars represent ±SEM (b, c). n = 5 (I130T mice) and 6 (G60S mice)

Fig. 6

Cx26 was localized to intracellular compartments of basal urothelial cells of mutant and wild-type (Wt) mice. Immunolabeling revealed that Cx26 was localized to intracellular compartments of the basal urothelium cells in all mice (a, b). Uro urothelium, LP lamina propria. Red indicates Cx26, and blue indicates Hoechst nuclear stain. Bar 50 μm