Action at a Distance in Gene Expression

Science Signaling  29 May 2012:
Vol. 5, Issue 226, pp. ec150
DOI: 10.1126/scisignal.2003262

Nkx3.1, a transcription factor critical for normal prostate development, acts as a tumor suppressor by inhibiting cell proliferation. It was assumed to suppress expression of proliferative genes within a cell, but Zhou et al. found that Nkx3.1 had a non–cell autonomous function. Human prostate epithelial cells transiently transfected with Nkx3.1 exhibited efficient growth inhibition despite poor transfection efficiency. Transwell assays separating transfected and untransfected cells showed that proliferation was inhibited in untransfected cells. Recombinant Nkx3.1 protein added exogenously to prostate epithelial cells inhibited growth in a dose-dependent manner. Either intracellular expression of Nkx3.1 or extracellular treatment of cells with recombinant protein lowered mRNA abundance of Nkx3.1 target genes and decreased luciferase reporter activity from their promoters. The authors developed a double-fluorescent labeling expression system using a construct encoding Nkx3.1 fused to a red fluorescent protein, then an internal ribosomal entry site, followed by a green fluorescent protein. Fluorescence microscopy of transfected cells showed yellow color in nuclei due to coexpression of both fluorescent proteins and green in the cytosol. Untransfected cells in the same culture dish had red nuclei, and the cytosol was not fluorescent, indicating the presence of Nkx3.1 but the absence of expression from the construct. The authors detected Nkx3.1 protein in cell-conditioned medium, normal mouse prostatic fluids, and mouse urine by Western blot with an antibody that recognizes Nkx3.1. Cells lines not derived from the prostate (nonprostatic), which do not have endogenous Nkx3.1, were transfected with either wild-type Nkx3.1 or Nkx3.1 harboring a point mutation associated with prostate cancer and cocultured with prostatic cells. Transwell assays showed inhibition of proliferation of prostatic cells when cultured with the cells expressing the wild type, but not with those expressing the mutant. Quantitative PCR specific for human mRNA showed suppression of Nkx3.1 target gene expression when the wild-type Nkx3.1 was expressed but not when the mutant was expressed. Chromatin immunoprecipitation assays performed with cocultures of nonprostatic cells expressing tagged Nkx3.1 and prostatic cells showed that Nkx3.1was bound to target genes in prostatic cells only in the cell cocultured with those expressing wild-type Nkx3.1, not with those expressing the mutant. The prostate cancer–associated mutant Nkx3.1 was not detected in cell-conditioned medium from prostatic cells expressing this mutant, suggesting that this mutation prevents its secretion. Thus, Nkx3.1 appears to be a secreted transcription factor that acts non–cell autonomously to inhibit gene expression. A prostate cancer–associated mutation in Nkx3.1 may diminish its tumor-suppression activity by preventing its secretion and transfer to adjacent cells.

J. Zhou, L. Qin, J. C.-Y. Tien, L. Gao, X. Chen, F. Wang, J.-T. Hsieh, J. Xu, Nkx3.1 functions as para-transcription factor to regulate gene expression and cell proliferation in non-cell autonomous manner. J. Biol. Chem. 287, 17248–17256 (2012). [Abstract] [Full Text]