Lam M H, Olsen S L, Rankin W A, Ho P W, Martin T J, Gillespie M T, Moseley J M
St. Vincent's Institute of Medical Research, University of Melbourne, Victoria, Australia.
J Cell Physiol. 1997 Dec;173(3):433-46. doi: 10.1002/(SICI)1097-4652(199712)173:3<433::AID-JCP16>3.0.CO;2-C.
Parathyroid hormone-related protein (PTHrP) is highly expressed in normal skin keratinocytes, and its involvement in growth and differentiation processes in these cells has been implicated by several lines of evidence which include the use of antisense PTHrP (Kaiser et al., 1994, Mol. Endocrinol., 8:139-147). In this study, we have investigated whether PTHrP expression and its subcellular localization is linked to cell cycle progression in a human keratinocyte cell line (HaCat), which constitutively expresses and secretes PTHrP. PTHrP mRNA and immunoreactive PTHrP were assessed in asynchronous dividing cells and in cells blocked at G1 or G2 + M phases of the cell cycle using several different protocols. The response of PTHrP mRNA expression was examined following readdition of serum in the continued presence of cycle blockers, and after release from cell cycle block, or from cell synchronization by serum deprivation. PTHrP expression was greatest in actively dividing cells when cells were in S and G2 + M phases of the cell cycle and were lowest in quiescent G1 cells. Most notable were the high levels of PTHrP mRNA and protein in cells at G2 + 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 PTHrP in cell division in keratinocytes. In asynchronously growing cells, PTHrP expression fell as cells became confluent at a time when cell growth is inhibited and cells begin to differentiate. Mitogen stimulation of HaCaT cells resulted in a rapid increase in PTHrP mRNA expression, but was dependent upon cells being in the G1 phase of the cell cycle. Cells blocked in G1 responded to mitogen both in the continued presence of aphidicolin or when released from block. Cells blocked at G2 + M with colcemid expressed high levels of PTHrP mRNA and protein, and PTHrP mRNA did not respond further to mitogen in the continued presence of blocker. However, in cells released from block at G2 + M by addition of serum, an increase in PTHrP expression was seen coincident with the progression of cells into G1. In contrast, in a squamous 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. The results of this study suggest that PTHrP may have two roles in the cell cycle; one in G1 in response to mitogen, and a second at cell division when its expression is high and it is relocated from the nucleolus to the cytoplasm.
甲状旁腺激素相关蛋白（PTHrP）在正常皮肤角质形成细胞中高度表达，多条证据表明其参与了这些细胞的生长和分化过程，其中包括使用反义PTHrP（Kaiser等人，1994年，《分子内分泌学》，8:139 - 147）。在本研究中，我们调查了PTHrP的表达及其亚细胞定位是否与人类角质形成细胞系（HaCat）的细胞周期进程相关，该细胞系组成性表达并分泌PTHrP。使用几种不同的方案，在异步分裂细胞以及在细胞周期的G1期或G2 + M期被阻断的细胞中评估了PTHrP mRNA和免疫反应性PTHrP。在持续存在周期阻滞剂的情况下重新添加血清后，以及从细胞周期阻滞或血清剥夺导致的细胞同步化中释放后，检测了PTHrP mRNA表达的反应。当细胞处于细胞周期的S期和G2 + M期且处于活跃分裂状态时，PTHrP表达最高，而在静止的G1期细胞中最低。最值得注意的是，在细胞周期G2 + M期分裂的细胞中PTHrP mRNA和蛋白质水平很高。此外，PTHrP在静止细胞中定位于核仁，但在细胞活跃分裂时重新分布到细胞质中。综上所述，这些结果支持PTHrP在角质形成细胞的细胞分裂中发挥作用。在异步生长的细胞中，当细胞生长受到抑制且开始分化时，随着细胞汇合，PTHrP表达下降。对HaCaT细胞的丝裂原刺激导致PTHrP mRNA表达迅速增加，但这取决于细胞处于细胞周期的G1期。在持续存在阿非科林的情况下或从阻滞中释放后，G1期被阻断的细胞对丝裂原作出反应。用秋水仙酰胺阻断在G2 + M期的细胞表达高水平的PTHrP mRNA和蛋白质，并且在持续存在阻滞剂的情况下，PTHrP mRNA对丝裂原不再有进一步反应。然而，在通过添加血清从G2 + M期阻滞中释放的细胞中，随着细胞进入G1期，可见PTHrP表达增加。相比之下，在鳞状癌细胞系（COLO16）中，基础PTHrP表达很高，并且在细胞周期中或通过细胞周期阻滞均未改变，这与其在恶性细胞中表达失调的关联一致。本研究结果表明，PTHrP在细胞周期中可能具有两个作用；一个是在G1期响应丝裂原，另一个是在细胞分裂时，此时其表达很高且从核仁重新定位到细胞质中。
