Asymmetrical cell division and differentiation are not dependent upon stratification in a corneal epithelial cell...

To determine whether asymmetrical cell division takes place during growth and differentiation of corneal epithelial cells, we analyzed the expression of some proteins required for the correct execution of the asymmetric division in cultured RCE1-(5T5) cells, which mimic the differentiation of corneal epithelial cells. RT-PCR and immunostaining showed that Par-3, LGN (GPSM2), NuMA, and the mammalian homolog of inscuteable (Insc) are expressed by the cultured cells. Semi-quantitative RT-PCR demonstrated that Insc mRNA levels were stable throughout the experiment. Conversely, LGN and NuMA mRNAs increased slightly and steadily in proliferative cells, reaching a peak of about 20% above basal levels when cells were confluent. At later times, LGN and NuMA mRNAs decreased to become barely detectable when cells organized into a four-layered epithelium and expressed terminal phenotype as indicated by the highest expression of LDH-H mRNA. Cultivation under low Ca2+ conditions (0.09 mM) reduced about 50% Insc mRNA expression both in proliferating and confluent cultures, but did not affect the levels of LGN and NuMA mRNAs. Hence, asymmetric cell division seems to take place with a lower frequency in cells grown with low Ca2+ concentrations, in spite of the absence of stratification. Immunostaining experiments raise the possibility of an interaction between k3/K12 keratin cytoskeleton and Par-3. The results show for the first time the coordination between the expression of corneal epithelial cell differentiation and the expression of cell polarity machinery. They also suggest that asymmetric division does not depend on stratification; instead, it seems to be part of the differentiation program.