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In JoVE (1)

Other Publications (6)

Articles by Steven Kregel in JoVE

 JoVE Clinical and Translational Medicine

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

1Department of Surgery, Section of Urology, University of Chicago, 2Committee on Cancer Biology, University of Chicago


JoVE 50327

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

Other articles by Steven Kregel on PubMed

Downregulation of Smooth Muscle Alpha-actin Expression by Bacterial Lipopolysaccharide

Smooth muscle alpha-actin (SMA) is a cytoskeletal protein characteristic to vascular smooth muscle cells (VSMC), and it serves to facilitate cell contraction and migration. Bacterial lipopolysaccharide (LPS), a major mediator of septic shock secondary to infection, is known to directly affect VSMC. The objective of this study was to investigate the effect of LPS on the expression levels of SMA in VSMC.

Regulation of Smad-mediated Gene Transcription by RGS3

Regulator of G protein signaling (RGS) proteins are united into a family by the presence of the homologous RGS domain that binds the alpha subunits of heterotrimeric G proteins and accelerates their GTPase activity. A member of this family, RGS3 regulates the signaling mediated by G(q) and G(i) proteins by binding the corresponding Galpha subunits. Here we show that RGS3 interacts with the novel partners Smad2, Smad3, and Smad4-the transcription factors that are activated through a transforming growth factor-beta (TGF-beta) receptor signaling. This interaction is mediated by the region of RGS3 outside of the RGS domain and by Smad's Mad homology 2 domain. Overexpression of RGS3 results in inhibition of Smad-mediated gene transcription. RGS3 does not affect TGF-beta-induced Smad phosphorylation, but it prevents heteromerization of Smad3 with Smad4, which is required for transcriptional activity of Smads. This translates to functional inhibition of TGF-beta-induced myofibroblast differentiation by RGS3. In conclusion, this study identifies a novel, noncanonical role of RGS3 in regulation of TGF-beta signaling through its interaction with Smads and interfering with Smad heteromerization.

Critical Role of Serum Response Factor in Pulmonary Myofibroblast Differentiation Induced by TGF-beta

Transforming growth factor-beta (TGF-beta) is a cytokine implicated in wound healing and in the pathogenesis of pulmonary fibrosis. TGF-beta stimulates myofibroblast differentiation characterized by expression of contractile smooth muscle (SM)-specific proteins such as SM-alpha-actin. In the present study, we examined the role of serum response factor (SRF) in the mechanism of TGF-beta-induced pulmonary myofibroblast differentiation of human lung fibroblasts (HLF). TGF-beta stimulated SM-alpha-actin expression in HLF, which paralleled with a profound induction of SRF expression and activity. Inhibition of SRF by the pharmacologic SRF inhibitor (CCG-1423), or via adenovirus-mediated transduction of SRF short hairpin RNA (shSRF), blocked the expression of both SRF and SM-alpha-actin in response to TGF-beta without affecting Smad-mediated signaling of TGF-beta. However, forced expression of SRF on its own did not promote SM-alpha-actin expression, whereas expression of the constitutively transactivated SRF fusion protein (SRF-VP16) was sufficient to induce SM-alpha-actin expression, suggesting that both expression and transactivation of SRF are important. Activation of protein kinase A (PKA) by forskolin or iloprost resulted in a significant inhibition of SM-alpha-actin expression induced by TGF-beta, and this was associated with inhibition of both SRF expression and activity, but not of Smad-mediated gene transcription. In summary, this is the first direct demonstration that TGF-beta-induced pulmonary myofibroblast differentiation is mediated by SRF, and that inhibition of myofibroblast differentiation by PKA occurs through down-regulation of SRF expression levels and SRF activity, independent of Smad signaling.

Retinoic Acid Fails to Induce Cell Cycle Arrest with Myogenic Differentiation in Rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Current treatment strategies do not cure most children with recurrent or high-risk disease, underlying the need for novel therapeutic approaches. Retinoic acid has been shown to induce differentiation in a variety of cells including skeletal myoblasts and neuroblasts. In the setting of minimal residual disease, retinoic acid improves survival in neuroblastoma, another poorly differentiated childhood tumor. Whether such an approach is useful for rhabdomyosarcoma has not yet been investigated. Several in vitro studies have demonstrated an appreciable effect of retinoic acid on human RMS cellular proliferation and differentiation.

Growth Kinetics of CD133-positive Prostate Cancer Cells

In the adult human prostate CD133 expression is thought to mark rare prostate epithelial stem cells and malignant tumor stem/initiating cells. Such putative stem cell populations are thought to proliferate slowly, but possess unlimited proliferative potential. Based on this, we hypothesized that CD133(pos) prostate cancer cells proliferate slower than CD133(neg) cells.

Sox2 is an Androgen Receptor-repressed Gene That Promotes Castration-resistant Prostate Cancer

Despite advances in detection and therapy, castration-resistant prostate cancer continues to be a major clinical problem. The aberrant activity of stem cell pathways, and their regulation by the Androgen Receptor (AR), has the potential to provide insight into novel mechanisms and pathways to prevent and treat advanced, castrate-resistant prostate cancers. To this end, we investigated the role of the embryonic stem cell regulator Sox2 [SRY (sex determining region Y)-box 2] in normal and malignant prostate epithelial cells. In the normal prostate, Sox2 is expressed in a portion of basal epithelial cells. Prostate tumors were either Sox2-positive or Sox2-negative, with the percentage of Sox2-positive tumors increasing with Gleason Score and metastases. In the castration-resistant prostate cancer cell line CWR-R1, endogenous expression of Sox2 was repressed by AR signaling, and AR chromatin-IP shows that AR binds the enhancer element within the Sox2 promoter. Likewise, in normal prostate epithelial cells and human embryonic stem cells, increased AR signaling also decreases Sox2 expression. Resistance to the anti-androgen MDV3100 results in a marked increase in Sox2 expression within three prostate cancer cell lines, and in the castration-sensitive LAPC-4 prostate cancer cell line ectopic expression of Sox2 was sufficient to promote castration-resistant tumor formation. Loss of Sox2 expression in the castration-resistant CWR-R1 prostate cancer cell line inhibited cell growth. Up-regulation of Sox2 was not associated with increased CD133 expression but was associated with increased FGF5 (Fibroblast Growth Factor 5) expression. These data propose a model of elevated Sox2 expression due to loss of AR-mediated repression during castration, and consequent castration-resistance via mechanisms not involving induction of canonical embryonic stem cell pathways.

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