Figure 4.

Syndecan regulation of calcium and cell adhesion through TRPC4 in epithelial cells. (A) Syndecan expression in HaCaT cells was analyzed by quantitative RT-PCR. Syndecan-1 and -4 were expressed highly in contrast to syndecan-2 and -3. (B) Single knockdown of syndecan-1 or -4 in HaCaT epidermal cells does not affect cytosolic calcium levels, whereas reduction of both proteoglycans yields reduced cytosolic calcium. n = 26–56. (C) TRPC4 associates with syndecan-1 and -4. Specific syndecan antibodies were used to cluster and precipitate complexes that were probed for TRPC4. Normal rat IgG was used as a control. (D) Silencing TRPC4 in HaCat cells reduces cytosolic calcium similar to that of double syndecan-1/4 knockdown. Combining siRNA for the syndecans and TRPC4 together does not further reduce cytosolic calcium. n > 40 cells in each case. (E) Differentiation markers keratin 1 and involucrin, but not basal keratin 5, mRNA levels were decreased on reduction of syndecan-1 and -4 or TRPC4 (n = 3). (F and G) P-cadherin immunocytochemistry in HaCat cells silenced for syndecan-1 and -4 showed an increase at adherens junctions (arrowheads) after 4 h of culture in high calcium medium to stimulate differentiation. Similar effects are seen in cells silenced for TRPC4 expression or TRPC4 silenced in a syndecan null background. Data are shown from five replicates. (H) wt newborn murine skin contains no discernible P-cadherin in interfollicular epidermis, but it is strongly expressed in the basal and spinous layers of the syndecan-1/4 double KO tissue. (I) Keratin 10 is strictly suprabasal in wt mouse (left) and rat skin (right), but keratin 10–positive cytoplasmic extensions to the basement membrane (marked in red) are present in the syndecan null and TRPC4 null epidermis (arrowheads). Insets in H and I show higher magnification images of boxed areas. Bars, 10 µm. (J) Electron micrographs of skin samples from two different tissue blocks from neonate wt mouse and rat skin, s1s4 double KO mouse, and TRPC4 KO rat skin were analyzed (examples shown in Fig. S3 E). Total discrete cell profiles in contact with the dermal–epidermal junction were quantitated. A total of 24 frames (86 cells) for mouse wt, 70 frames (454 cells) for s1s4 KO mouse, 127 frames (148 cells) for wt rat, and 82 frames (208 cells) for TRPC4 KO rat were quantitated. Statistical analysis was performed and group differences were determined using unpaired t test. ****, P < 0.0001 for both sets of null animals versus their respective wt controls. See also Fig. S3. Data are the mean ± SEM. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; unpaired t test.