In our previous study, we demonstrate that CD44, once engaged, is internalized and translocated to the nucleus, where it binds to various promoters leading to cell fate change (accelerating cell http://www.selleckchem.com/products/mek162.html proliferation) through transcriptional reprogramming (Lee et al, 2009). As shown in Figure 3A, ectopic expression of wild-type CD44s but not the CD44(NLS) mutant (which cannot translocate into the nucleus, as proved by immunofluorescence staining) conferred increased sphere size, suggesting that localization of CD44 in the nucleus is an important aspect of its growth-stimulating function. Moreover, in the nucleus, the acetylated-STAT3 dimer remains associated with CD44 through the N-terminal region encoded by C3 and binds to various promoters, leading to increased cell proliferation (Lee et al, 2009).
Consistent with our previous report, HT29/CD44?/CD44sC4�CC10 (62.5��3.5 ��m) and HT29/CD44?/CD44sC6�CC10 (54.1��5.3 ��m) (which are defective in association with STAT3) significantly decreased the size of spheres in comparison to spheres expressing wild-type CD44s (137.2��6.1 ��m), CD44sC3�CC10 (128��4.9 ��m) and CD44s��C4�CC7 (132.9��6.7 ��m) (Figure 3A), demonstrating that nuclear CD44/STAT3 is crucial for sphere growth. Next, we further tested whether cells cultured in suspension could enhance CD44 and STAT3 translocation into the nucleus. As shown in Figure 3B, cells cultured in suspension promoted CD44 and STAT3 nuclear translocation in a time-dependent manner. In contrast, the CD44(NLS) mutant failed to enter the nucleus.
To corroborate that the bipartite NLS is essential for the nuclear localization of CD44 protein, a Ran GTPase mutant Ran(Q69L) (Macara, 2001) was ectopically expressed in HT29/CD44?/CD44-myc cells. Figure 3B shows that overexpression of Ran(Q69L) completely blocked CD44 and STAT3 nuclear import after the suspension culture. According to western blotting, all of the CD44 N-terminal deletion mutants in HT29/CD44?/CD44-myc spheres could translocate into the nucleus except for the CD44s(NLS) mutant, and the amount of nuclear CD44 in spheres was higher than in cells maintained as GSK-3 subconfluent monolayers (WT/AD) (Figure 3C). Co-immunoprecipitation further revealed that STAT3 and p300 were in complex with CD44 in HT29/CD44?/CD44-myc spheres (Figure 3C). The similar results were also shown in HCT-116 and DLD-1 cells (Supplementary Figure S4).