Abstract 1267

c-Kit, the cytokine receptor that mediates SCF-induced cell proliferation, has recently been found to trigger cell polarity establishment in mouse HSPC. SCF stimulation of cultured bone marrow cells induces polarization of c-Kit and the aPKC-Par6-Par3 cell polarity machinery to one pole, followed by asymmetric division into dissimilar daughter cells. The SCF-induced redistribution of proteins in HSPC resembles that of polarized epithelial cells, in which cell surface proteins selectively traffic to either an apical or basolateral domain. We hypothesize that c-Kit might behave like an “apical” protein in this context. To test this hypothesis, we retrovirally transduced c-Kit constructs into Madin-Darby Canine Kidney (MDCK) cells, an epithelial cell line that forms polarized cysts when cultured in collagen gel. Each polarized MDCK cyst forms an apical surface facing inside, and a basolateral surface facing outside. When expressed in these cysts, a wild-type c-Kit construct traveled only to the apical surface and never to the basolateral surface, in agreement with our hypothesis. We next examined the distribution of c-Kit-BL, a c-Kit mutant that contained in its cytoplasmic domain a basolateral-trafficking (BL) signal sequence derived from TGFβ receptor. The c-Kit-BL mutant lost its apical trafficking preference and distributed instead to the BL surface, suggesting that the apical distribution of c-Kit could be blocked by an aberrant trafficking signal. We then examined the distribution of c-Kit expressed in MDCK cells cultured in liquid suspension. Under this condition, MDCK cells aggregated to form inside-out spheres, with an apical outer surface and a BL inner surface. Along with the polarity reversal of the spheres, the distribution patterns of c-Kit and c-Kit-BL were also reversed. Embedding the inside-out spheres in collagen gel re-established the original polarity, and the c-Kit constructs regained their original distributions. We then tested the behavior of c-Kit-BL-A531G and c-Kit-BL-Q532A, revertant and pseudo-revertant of c-Kit-BL, in which a critical residue (A531G) or non-critical residue (Q532A) of the BL signal sequence was mutated. We found that c-Kit-BL-A531G re-established apical distribution, whereas c-Kit-BL-Q532A remained at the basolateral surface. These results show that c-Kit behaved like an apical protein in the milieu of polarized epithelial cells and followed similar protein trafficking rules. We then asked if the apical trafficking capacity of c-Kit correlates with its ability to mediate SCF-induced proliferation of blood cells. We transduced c-Kit or c-Kit-BL into 32D cells, a mouse myelomonocytic cell line that normally does not express c-Kit and requires stimulation with hematopoietic growth factors to proliferate. Untransduced 32D cells proliferated only in response to IL3 stimulation, whereas c-Kit-transduced 32D cells also proliferated upon SCF simulation. By contrast, c-Kit-BL-transduced 32D cells behaved like the parental 32D cells and did not proliferate upon SCF stimulation, suggesting that a c-Kit mutant deficient in apical trafficking could not mediate proliferation. We next examined the effect of c-Kit or c-Kit-BL expression on polarization and proliferation of primary HSPC. We transduced mouse bone marrow cells to express either c-Kit or c-Kit-BL and examined for SCF-induced polarization. 76–96% of c-Kit-transduced HPSC exhibited polarization of markers Sca1, aPKC, β-catenin and annexin 2 upon SCF stimulation, whereas only 16–26% of c-Kit-BL-transduced cells underwent such polarization. We then cultured transduced bone marrow cells in methylcellulose in the presence of SCF to form colonies. c-Kit-transduced cells gave rise to discrete colonies but c-Kit-BL-transduced cells failed to produce any colonies, indicating that the c-Kit-BL mutant dominantly interfered with the ability of the endogenous c-Kit to promote proliferation. In summary, our data show that c-Kit trafficked selectively to an “apical” domain in epithelial cells and HSPC. A c-Kit mutant defective in the apical trafficking not only failed to induce blood cell proliferation but also suppressed the proliferative function of the endogenous c-Kit. Further delineation of the mechanism of apical trafficking of c-Kit should help elucidate the relationship between the cell polarity machinery and the regulation of HSPC proliferation.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.

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