Expression of a releasable form of annexin II by human keratinocytes

Abstract

Annexin II is a multifunctional calcium-dependent phospholipid binding protein whose presence in epidermis has previously been reported. However, like other members of annexin family, annexin II has been regarded as either an intracellular protein or associated with the cellular membrane. Here, we report the presence of a releasable annexin II and p11, two monomers of annexin II tetramer, in keratinocyte-conditioned medium (KCM). Proteins present in KCM were fractionated on a gel filtration column and following further evaluation, a releasable protein with apparent MW of 36 kDa was identified. Further characterization identified this protein as the p36 monomer of annexin II tetramer. The phospho-tyrosine antibody did not visualize this protein as the phosphorylated form of p36. Several experiments were conducted to examine whether this protein is soluble or associated with keratinocyte cell membranes in the conditioned medium. A centrifugation of conditioned medium was not able to bring this protein down into the pellet. Surprisingly, the results of Western analysis identified p36 and p11, two monomers of the annexin II tetramer, in conditioned medium derived from either keratinocytes cultured alone or keratinocytes co-cultured with fibroblasts. In contrast to the keratinocyte-conditioned medium in which annexin II was easily detectable, both monomers were barely detectable in conditioned medium collected from dermal fibroblasts. This finding was in contrast to the cell lysates in which p36 was detectable in both keratinocytes and fibroblasts. However, the amount of this protein was markedly higher in keratinocyte lysate relative to that of dermal fibroblasts. Conditioned medium derived from keratinocyte established from adult showed a higher level of annexin II compared to that of keratinocytes established from newborn babies. The expression of p11 seems to increase with differentiation of keratinocytes derived from either adult or newborn skin samples. When the site of annexin synthesis in human skin was examined by immunohistochemical staining, the antibody for p36 localized the annexin to the keratinocyte cell members in the basal and suprabasal keratinocytes. In conclusion, Western blot detection of both p36 and p11 in conditioned medium from skin cells revealed that human keratinocytes, but not fibroblasts, express a releasable monomer form of annexin II which is regulated by differentiation status of keratinocytes. This finding is consistent with the localization of annexin II detected by immunohistochemical staining.