In situ lineage tracking of human prostatic epithelial stem cell fate reveals a common clonal origin for basal and luminal cells.

Stem cells accumulate mitochondrial DNA (mtDNA) mutations resulting in an observable respiratory chain defect in their progeny, allowing the mapping of stem cell fate. There is considerable uncertainty in prostate epithelial biology where both basal and luminal stem cells have been described, and in this study the clonal relationships within the human prostate epithelial cell layers were explored by tracing stem cell fate. Fresh-frozen and formalin-fixed histologically-benign prostate samples from 35 patients were studied using sequential cytochrome c oxidase (COX)/succinate dehydrogenase (SDH) enzyme histochemistry and COX subunit I immunofluorescence to identify areas of respiratory chain deficiency; mtDNA mutations were identified by whole mitochondrial genome sequencing of laser-captured areas. We demonstrated that cells with respiratory chain defects due to somatic mtDNA point mutations were present in prostate epithelia and clonally expand in acini. Lineage tracing revealed distinct patterning of stem cell fate with mtDNA mutations spreading throughout the whole acinus or, more commonly, present as mosaic acinar defects. This suggests that individual acini are typically generated from multiple stem cells, and the presence of whole COX-deficient acini suggests that a single stem cell can also generate an entire branching acinar subunit of the gland. Significantly, a common clonal origin for basal, luminal and neuroendocrine cells is demonstrated, helping to resolve a key area of debate in human prostate stem cell biology.

Phenotypic modulation of human urinary tract stroma-derived fibroblasts by transforming growth factor beta3.

OBJECTIVES: Animal models have described critical roles for transforming growth factor beta (TGFbeta) isoforms in modulating urinary tract stroma phenotype. TGFbeta3 is of particular interest because it may regulate TGFbeta1 and TGFbeta2 expression, but its modulatory affect has not been so well characterized in human cells. In this study, we aim to determine whether TGFbeta3 treatment induced differentiation of human urinary tract stroma-derived fibroblasts to a smooth muscle-like phenotype. METHODS: We established cultures of human urinary tract stroma-derived fibroblasts and studied the effects of TGFbeta3 treatment using proliferation assays, cell cycle analysis, immunocytochemistry, and Western blotting for expression of differentiation marker and downstream regulators, and fura-2 fluorescence to study the effects on intracellular calcium. RESULTS: TGFbeta3 treatment induced proliferation that peaked at 72 hours, followed by enhanced expression of alpha-smooth muscle actin (alpha-SMA) with a maximal 3.4-fold increase at 168 hours. TGFbeta3 treatment decreased resting [Ca(2+)](i) by 70% and caused a 95% decrease in stimulated internal Ca(2+) release regulated by the sarcoplasmic/endoplasmic calcium-ATPase pump. These effects were associated with upregulation of nuclear activator of T cells -1 (NFAT), a known regulator of cell differentiation. CONCLUSIONS: TGFbeta3 treatment causes a time-specific response in the presence of serum, whereby fibroblasts initially proliferate and subsequently differentiate to a smooth muscle-like phenotype. This sequence was associated with stabilization of [Ca(2+)](i) stores, suggesting a role in the induction of hyperplasia and reduction of contractility; phenomena associated with a number of urinary tract pathologies.

The scaffolding protein RACK1 interacts with androgen receptor and promotes cross-talk through a protein kinase C signaling pathway.

The androgen receptor (AR), a member of the nuclear hormone receptor superfamily, functions as a ligand-dependent transcription factor that regulates genes involved in cell proliferation and differentiation. Using a C-terminal region of the human AR in a yeast two-hybrid screen, we have identified RACK1 (receptor for activated C kinase-1) as an AR-interacting protein. In this report we found that RACK1, which was previously shown to be a protein kinase C (PKC)-anchoring protein that determines the localization of activated PKCbetaII isoform, facilitates ligand-independent AR nuclear translocation upon PKC activation by indolactam V. We also observed RACK1 to suppress ligand-dependent and -independent AR transactivation through PKC activation. In chromatin immunoprecipitation assays, we demonstrate a decrease in AR recruitment to the AR-responsive prostate-specific antigen (PSA) promoter following stimulation of PKC. Furthermore, prolonged exposure to indolactam V, a PKC activator, caused a reduction in PSA mRNA expression in prostate cancer LNCaP cells. Finally, we found PKC activation to have a repressive effect on AR and PSA protein expression in androgen-treated LNCaP cells. Our data suggest that RACK1 may function as a scaffold for the association and modification of AR by PKC enabling translocation of AR to the nucleus but rendering AR unable to activate transcription of its target genes.