PTHRP AND CELL-DIVISION - EXPRESSION AND LOCALIZATION OF PTHRP IN A KERATINOCYTE CELL-LINE (HACAT) DURING THE CELL-CYCLE

Parathyroid hormone-related protein (PTHrP) is highly expressed in nor mal skin keratinocytes, and its involvement in growth and differentiat ion processes in these cells has been implicated by several lines of e vidence which include the use of antisense PTHrP (Kaiser et al., 1994, Mel. Endocrinol., 8:139-147). In this study, we have investigated whe ther PTHrP expression and its subcellular localization is linked to ce ll cycle progression in a human keratinocyte cell line (HaCaT), which constitutively expresses and secretes PTHrP. PTHrP mRNA and immunoreac tive PTHrP were assessed in asynchronous dividing cells and in cells b locked at G(1) or G(2) + M phases of the cell cycle using several diff erent protocols. The response of PTHrP mRNA expression was examined fo llowing readdition of serum in the continued presence of cycle blocker s, and after release from cell cycle block, or from cell synchronizati on by serum deprivation. PTHrP expression was greatest in actively div iding cells when cells were in S and G(2) + M phases of the cell cycle and were lowest in quiescent G(1) cells. Most notable were the high l evels of PTHrP mRNA and protein in cells at G(2) + M phase of the cell cycle at division. Furthermore, PTHrP was localized to the nucleolus in quiescent cells, but redistributed to the cytoplasm when cells were ;actively dividing. Taken together, these results support a role for P THrP in cell division in keratinocytes. In asynchronously growing cell s, PTHrP expression fell as cells became confluent at a time when cell growth is inhibited and cells begin to differentiate. Mitogen stimula tion of HaCaT cells resulted in a rapid increase in PTHrP mRNA express ion, but was dependent upon cells being in the G(1) phase of the cell cycle. Cells blocked in G(1) responded to mitogen both in the continue d presence of aphidicolin or when released from block. Cells blocked a t G(2) + M With colcemid expressed high levels of PTHrP mRNA and prote in, and PTHrP mRNA did not respond further to mitogen in the continued presence of blocker. However, in cells released from block at G(2) M by addition of serum, an increase in PTHrP expression was seen coinc ident with the progression of cells into G(1). In contrast, in a squam ous cancer cell line (COLO16), basal PTHrP expression was high and was not altered during the cell cycle or by cell cycle block, consistent with association of its dysregulated expression in malignant cells. Th e results of this study suggest that PTHrP may have two roles in the c ell cycle; one in G(1) in response to mitogen, and a second at cell di vision when its expression is high and it is relocated from the nucleo lus to the cytoplasm. (C) 1997 Wiley-Liss, Inc.