The multidrug resistance P‐glycoprotein (P‐gp), which transports hydrophobic drugs out of cells, is also associated with volume‐activated chloride currents. It is not yet clear whether P‐gp is a channel itself, or whether it is a channel regulator. Activation of chloride currents by hypotonicity in cells expressing P‐gp was shown to be regulated by protein kinase C (PKC). HeLa cells exhibited volume‐activated chloride currents indistinguishable from those obtained in P‐gp‐expressing cells except that they were insensitive to PKC. HeLa cells did not express detectable P‐gp but, following transient transfection with cDNA encoding P‐gp, the volume‐activated channels acquired PKC regulation. PKC regulation was abolished when serine/threonine residues in the consensus phosphorylation sites of the linker region of P‐gp were replaced with alanine. Replacement of these residues with glutamate, in order to mimic the charge of the phosphorylated protein, also mimicked the effects of PKC on channel activation. These data demonstrate that PKC‐mediated phosphorylation of P‐gp regulates the activity of an endogenous chloride channel and thus indicate that P‐gp is a channel regulator.

PTHrP is secreted by breast cancer cells in vivo and in vitro. In the breast cancer cell line MCF-7, PTHrP overexpression is associated with increased mitogenesis. We used this cell line to study the mechanism for the proliferative effects of PTHrP. Clonal MCF-7 lines were established overexpressing wild-type PTHrP or PTHrP mutated in the nuclear localization signal (NLS). Mutation of the NLS negated the proliferative effects and nuclear trafficking of PTHrP, indicating that increased mitogenesis is mediated via an intracrine pathway. Cells overexpressing wild-type PTHrP were enriched in G2 + M stage of the cell cycle compared with cells overexpressing NLS-mutated PTHrP, indicating an intracrine role for PTHrP in cell cycle regulation. Wild-type PTHrP also protected MCF-7 cells from serum starvation-induced apoptosis. Cells overexpressing wild-type PTHrP showed significantly greater cell survival than cells overexpressing NLS-mutated PTHrP. The ratios of the apoptosis-regulating proteins Bcl-2 to Bax and Bcl-x(L) to Bax were higher in cells overexpressing wild-type, but not NLS-mutated, PTHrP compared with control cells. These findings suggest that the proliferative effects of PTHrP in breast cancer cells are mediated through regulation of the cell cycle and apoptosis, and that controlling PTHrP production in breast cancer may be therapeutically useful.

(8), and other cellular and physiological processes. Many of the actions of 1,25-D are mediated by an intracellular receptor, the vitamin D receptor (VDR), a member of the steroid/thyroid receptor superfamily of ligandregulated transcription factors (9, 10). The activity of VDR is dependent not only on the concentration of receptor and hormone but also on its heterodimer partner, retinoid X receptor (RXR), and the coactivator proteins that bind to the VDR and facilitate transcription of target genes (11).In addition to its actions as a modulator of transcription through activation of the VDR, 1,25-D can rapidly activate cell signaling cascades independent of a requirement for transcription (12-14). The means by which 1,25-D induces these changes has not been fully elucidated. Rapid activation of extracellular signal-regulated kinases, ERK1/ERK2 in NB4 promyelocytic leukemia cells can be induced not only by 1,25-D, but also by analogs that are unable to activate VDR, suggesting the possibility of a separate receptor (15). Antibodies to a membrane protein identified by Nemere et al. (16) block the ability of 1,25-D to induce rapid calcium uptake and activation of PKC in cartilage cells. VDRϪ/Ϫ osteoblasts take up calcium and activate PKC similar to the wild-type osteoblasts, implicating proteins other than VDR in these actions (17). In contrast, Gniadecki (18) has described activation of ERK through 1,25-D-induced activation of Raf as a result of interactions between VDR and the adaptor protein Shc. VDR-null osteoblasts do not exhibit ion channel responses in response to 1,25-D (13) and Erben et al. (14) have reported that deletion of the VDR DNA binding domain also eliminates non-genomic responses. Thus some of the rapid actions of 1,25-D may be dependent upon VDR, whereas others are not.Nuclear receptor family members including VDR and RXR as well as many of their coactivators, are phosphoproteins whose activities are also regulated by cell signaling pathways (19 -27). Thus 1,25-D can modulate VDR activity both through direct binding to VDR as well as by altering the kinase activities within the cell (9,11,12,28). Although VDR has not been reported to be a substrate for ERK, RXR␣ (one of the three RXR isoforms) (29) is phosphorylated by ERK, as are some of the VDR coactivators including SRC-1 (30).To better understand the functional interactions between VDR and the ERK signaling pathway, we sought to determine whether 1,25-D activates ERK in the osteoblastic cell lines, MG-63 and MC3T3-E1, and to evaluate the effects of ERK on VDR activity. We found that 1,25-D rapidly induced ERK activity and that this activation persisted at 24 h in both cell lines. Surprisingly, the effects of ERK activation on VDR activity in the two cell lines were very different. Overexpression of Raf-1 (an upstream activator of ERK) reduced VDR activity in MC3T3-E1 cells, but stimulated activity in MG-63 cells.

No abstract