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First published online July 2, 2007
doi: 10.1242/10.1242/jcs.007443

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Polarity proteins PAR6 and aPKC regulate cell death through GSK-3 in 3D epithelial morphogenesis

¹ Departments of Anatomy, and Biochemistry and Biophysics, University of California School of Medicine, San Francisco, CA 94158, USA
² Department of Pediatrics, University of California School of Medicine, San Francisco, CA, USA

View larger version (32K): [in this window] [in a new window]   Fig. 1. PAR6N promotes apoptosis in MDCK cystogenesis. (A,B) Wild-type MDCK and Myc-tagged PAR6N-expressing stable cell lines grown on collagen for 7 days were fixed, permeabilized and immunostained for Myc (green), -catenin (red) and ZO-1 (white) (A) and for Myc (green), actin (red) and cleaved caspase-3 (white) (B). White arrow and arrowhead indicate dead cells in A. Nuclei are shown as blue in all figures. (C) The levels of apoptosis were measured using a cell death ELISA. Results are the mean±s.d. of three experiments. (D) Wild-type MDCK and PAR6N lysates isolated from cysts were prepared at different time points (day 5, 7 and 13) and then analyzed for activation of caspase-3 using cleaved caspase-3 and GAPDH antibodies (loading control). (E) PAR6N cysts were treated with either a carrier (DMSO) or caspase inhibitor, zVAD-fmk (50 µM) three times at day 0, 4 and 6 and immunostained for cleaved caspase-3 (red) at day 7, as described in A. Bars, 10 µm.

View larger version (29K): [in this window] [in a new window]   Fig. 2. aPKC and mLgl are mislocalized in PAR6N cells. (A) Wild-type MDCK and PAR6N cysts at day 7 were immunolabeled for aPKC (green), -catenin (red) and actin (white). (B) Wild-type MDCK and PAR6N cysts at day 7 were immunolabeled for mLgl (green). Bars, 10 µm.

View larger version (32K): [in this window] [in a new window]   Fig. 3. Reduced aPKC activity results in increased cell death in PAR6N cells. (A) aPKC activity and PAR6 expression levels were analyzed in wild-type MDCK and PAR6N cells by immunoblot using, respectively, phospho-aPKC, total-aPKC and PAR6B antibodies. The quantification of three independent experiments is depicted. *P<0.05. (B) MDCK cysts grown in collagen were treated with either the non-myristoylated (control) or myristoylated form of aPKC pseudosubstrate peptide, at the indicated concentrations, at day 4; aPKC pseudosubstrate-containing media were refreshed at day 6. Immunostaining with Ki-67 (green) and cleaved caspase-3 (red) were performed at day 7. Bar, 10 µm. (C) Percentage of cysts containing apoptotic cells was quantified by cleaved caspase-3 staining comparing control and aPKC inhibitor-treated cells. Results are the mean±s.d. of three experiments.

View larger version (21K): [in this window] [in a new window]   Fig. 4. aPKC inhibition by RNAi mimics the effect of PAR6N on apoptosis. (A) MDCK cells transfected with either an empty vector (control) or pSuper-aPKC RNAi were grown on Matrigel for 3 days and subjected to immunoblotting with total-aPKC (upper panel) and GAPDH (lower panel). (B) Control and aPKC RNAi MDCK cells grown on Matrigel were immunostained with aPKC (green; left), ZO-1 (white; middle) and cleaved caspase-3 (red; right). Bar, 10 µm. (C) Percentage of apoptotic cells was evaluated by cleaved caspase-3 staining comparing control and aPKC RNAi MDCK cells. Results are the mean±s.d. of three experiments.

View larger version (31K): [in this window] [in a new window]   Fig. 5. GSK-3 is hyperactivated in PAR6N cells and GSK-3 inhibitors suppress PAR6N-induced apoptosis. (A) Immunoblot of phospho-GSK-3 (Ser9) (upper panel) and total GSK-3 (lower panel) from wild-type MDCK and PAR6N total SDS-lysates. (B) Wild-type MDCK and PAR6N cells grown in collagen for 4 days were then cultured in the presence of 20 µM SB216763, 50 µM LiCl or vehicle (DMSO) and immunostained with Myc (green), cleaved caspase-3 (red) and actin (white) at day 7. Values shown at the bottom of each panel indicate the percentage of cysts lacking cleaved caspase-3 staining (mean±s.d. of three experiments). Bar, 10 µm.

View larger version (30K): [in this window] [in a new window]   Fig. 6. GSK-3 activity is crucial for apoptosis in MDCK cystogenesis. (A) Wild-type MDCK cells were cultured on Matrigel (72 hours) and infected with Ad-LacZ, Ad-HA-GSK-3 (S9A), Ad-HA-GSK-3 (K85M) for 24 hours, or not infected (MDCK control). Immunostaining analysis was performed with HA (green), cleaved caspase-3 (red) and actin (white). Bar, 10 µm. (B) The percentage of cysts containing apoptotic cells was quantified by cleaved caspase-3 staining. Results are the mean±s.d. of three experiments. *P<0.05 compared with either Ad-LacZ or Ad-HA-GSK-3 (K85M). (C) MDCK cysts infected with Ad-LacZ, Ad-HA-GSK-3 (S9A), or Ad-HA-GSK-3 (K85M) were subjected to immunoblot with -gal (green), HA (red) and GAPDH (red).

View larger version (25K): [in this window] [in a new window]   Fig. 7. Cell death induced by active GSK-3 is partially rescued by expression of aPKC. (A) Wild-type MDCK and GFP-aPKC cells were cultured on Matrigel (72 hours) and infected with Ad-HA-GSK-3 (S9A). Immunostaining analysis was performed with GFP (green), cleaved caspase-3 (red) and HA (white). (B) Wild-type MDCK and GFP-aPKC cells were cultured on Matrigel (72 hours) and infected with Ad-gal or Ad-HA-GSK-3 (S9A). The percentage of cysts containing apoptotic cells was quantified by cleaved caspase-3 staining. Results are the mean±s.d. of three experiments. (C) MDCK cysts grown in collagen were treated with either the non-myristoylated (control) or myristoylated form of aPKC pseudosubstrate peptide at 50 µM at day 4; aPKC pseudosubstrate-containing media were refreshed everyday. Immunostaining with aPKC (green) and phosphorylated GSK-3 (red) was performed at day 7. (D) Wild-type MDCK and GFP-aPKC-overexpressing MDCK cells were embedded into collagen and cultured for 7 days. Immunostaining with GFP-aPKC (green) and phosphorylated GSK-3 (white) was performed at day 7. Bars, 10 µm. (E) Wild-type MDCK and PAR6N lysates at day 7 were subjected to immunoblot of total- and phospho-JNK antibody.