Melanie J. Grubisha

Coactivators and nuclear receptor transactivation.

A variety of coregulator proteins serve as partners for nuclear receptors orchestrating the molecular events required for receptor‐dependent transcriptional regulation. Some coregulators directly interact with nuclear receptors and provide a platform for recruitment of other factors that provide distinct biochemical activities that influence transcriptional efficiency. Coregulators can influence chromatin structure and activity via direct modification of histone proteins or by facilitating ATP‐dependent chromatin remodeling. They also have the capacity to impact multiple steps in the transcription process including initiation, elongation, and mRNA splicing. Genetic analysis in humans and animal models are revealing the important cell and tissue‐type specific actions of nuclear receptor coregulators as well and their role in human physiology and disease. J. Cell. Biochem. 104: 1580–1586, 2008.

Hic-5 influences genomic and non-genomic actions of the androgen receptor in prostate myofibroblasts.

There is extensive knowledge of androgen receptor (AR) signaling in cancer cells, but less regarding androgen action in stromal cells of the tumor microenvironment. We report here the genome-wide effects of a stromal cell specific molecular adapter and AR coregulator, hydrogen peroxide-inducible gene 5 (Hic-5/TGFB1I1), on AR function in prostate myofibroblasts. Following androgen stimulation, Hic-5 rapidly translocates to the nucleus, coincident with increased phosphorylation of focal adhesion kinase. As a coregulator, Hic-5 acted to amplify or inhibit regulation of approximately 50% of AR target genes, affected androgen regulation of growth, cell adhesion, motility and invasion. These data suggest Hic-5 as a transferable adaptor between focal adhesions and the nucleus of prostate myofibroblasts, where it acts a key mediator of the specificity and sensitivity of AR signaling. We propose a model in which Hic-5 coordinates AR signaling with adhesion and extracellular matrix contacts to regulate cell behavior in the tumor microenvironment.

A local paracrine and endocrine network involving TGFβ, Cox-2, ROS, and estrogen receptor β influences reactive stromal cell regulation of prostate cancer cell motility.

The tumor microenvironment plays a critical role in supporting cancer cells particularly as they disengage from limitations on their growth and motility imposed by surrounding nonreactive stromal cells. We show here that stromal-derived androgenic precursors are metabolized by DU145 human prostate cancer (PCa) cells to generate ligands for estrogen receptor-β, which act to limit their motility through transcriptional regulation of E-cadherin. Although primary human PCa-associated fibroblasts and the human WPMY-1-reactive prostate stromal cell line maintain this inherent estrogen receptor (ER)β-dependent motility inhibitor activity, they are subverted by TGF-β1 pro-oxidant signals derived from cocultured DU145 PCa cells. Specifically, stromal-produced H(2)O(2), which requires Cox-2, acts as a second paracrine factor to inhibit ERβ activity in adjacent DU145 cells. Chromatin immunoprecipitation analysis reveals that ERβ recruitment to the E-cadherin promoter is inhibited when H(2)O(2) is present. Both neutralization of H(2)O(2) with catalase and prevention of its production by silencing Cox-2 expression in stromal cells restore the motility-suppression activity of stromal-derived ERβ ligand precursors. These data suggest that reactive stromal cells may still have a capacity to limit cancer cell motility through a local endocrine network but must be protected from pro-oxidant signals triggered by cancer cell-derived TGF-β1 to exhibit this cancer-suppressive function.

Local endocrine, paracrine and redox signaling networks impact estrogen and androgen crosstalk in the prostate cancer microenvironment

Androgen receptor (AR) signaling is essential for the initial development and progression of prostate cancer (PCa) as well as the growth and survival of castration-resistant tumors. However, AR action may be opposed by estrogen receptor beta (ERß) that responds to androgen metabolites produced in the prostate. The balance between the activity of these two receptors is not only influenced by the steroidogenic capacity of the prostatic microenvironment but also by its redox status and local paracrine signals such as transforming growth factor-beta (TGF-ß). In this review, we highlight the studies that revealed select roles for AR and ERß in distinct compartments of the prostate cancer microenvironment. We also discuss new work that identified stromal-epithelial crosstalk through TGF-ß1 signaling that drives the production of reactive oxygen species in stromal cells thereby selectively limiting the anti-tumor activity of ERß in cancer cells. Therefore, any new therapeutic approaches that seek to limit AR but enhance ERß activity in PCa, must take into account potential adaptive changes in the tumor microenvironment that utilize paracrine signals and altered redox balance to divert local androgen metabolites towards AR at the expense of ERß.

Age-dependent increase in Kalirin-9 and Kalirin-12 transcripts in human orbitofrontal cortex

KALRN (KAL) is a Rho GEF that is highly involved in regulation of the actin cytoskeleton within dendrites. There are several isoforms of the protein that arise from differential splicing of KALRNs 66 exons. KAL isoforms have different functions in development. For example, overexpression of the KAL9 and KAL12 isoforms induce dendritic elongation in early development. However, in mature neurons KAL9 overexpression reduces dendritic length, a phenotype also observed in normal human ageing. We therefore hypothesized that KAL9 would have increased expression with age, and undertook to evaluate the expression of individual KALRN exons throughout the adult lifespan. Postmortem human brain grey matter from Brodmanns area (BA) 11 and BA47 derived from a cohort of 209 individuals without psychiatric or neurodegenerative disease, ranging in age from 16 to 91 years, were analysed for KALRN expression by Affymetrix exon array. Analysis of the exon array data in an isoform‐specific manner, as well as confirmatory isoform‐specific qPCR studies, indicated that the longer KAL9 and KAL12 isoforms demonstrated a statistically significant, but modest, increase with age. The small magnitude of the age effect suggests that inter‐individual factors other than age likely contribute to a higher degree to KAL9 and KAL12 expression. In contrast to KAL9 and KAL12, global KALRN expression did not increase with age. Our work suggests that global measures of KALRN gene expression may be misleading and future studies should focus on isoform‐specific quantification.

A Schizophrenia-Linked KALRN Coding Variant Alters Neuron Morphology, Protein Function, and Transcript Stability

BACKGROUND Large-scale genetic studies have revealed that rare sequence variants, including single nucleotide variants (SNVs), in glutamatergic synaptic genes are enriched in schizophrenia patients. However, the majority are too rare to show any association with disease and have not been examined functionally. One such SNV, KALRN-P2255T, displays a penetrance that greatly exceeds that of previously identified schizophrenia-associated SNVs. Therefore, we sought to characterize its effects on the function of kalirin (Kal)-9, a dual Ras-related C3 botulinum toxin substrate 1 and Ras homologue gene family, member A (RhoA) guanine nucleotide exchange factor, upregulated in human schizophrenia brain tissue. METHODS Kal9 was overexpressed in primary rat cortical neurons or human embryonic kidney 293 (HEK293) cells. The effects of the P2255T variant on dendritic branching, dendritic spine morphology, protein and messenger RNA stability, and catalytic activity were examined. RESULTS Kal9-P2255T leads to diminished basal dendritic branching and dendritic spine size, compared with wild-type Kal9. The P2255T SNV directly affected Kal9 protein function, causing increased RhoA activation in HEK293 cells, but had no effect on Ras-related C3 botulinum toxin substrate 1 activation. Consistent with human postmortem findings, we found that Kal9-P2255T protein levels were higher than those of wild-type Kal9 in neurons. Increased messenger RNA stability was detected in HEK293 cells, indicating that this was the cause of the higher protein levels. When analyzed together, increased intrinsic RhoA guanine nucleotide exchange factor catalytic activity combined with increased messenger RNA expression led to net enhancement of RhoA activation, known to negatively impact neuronal morphology. CONCLUSIONS Taken together, our data reveal a novel mechanism for disease-associated SNVs and provide a platform for modeling morphological changes in mental disorders.

Opposing Effects of Cyclooxygenase-2 (COX-2) on Estrogen Receptor β (ERβ) Response to 5α-reductase Inhibition in Prostate Epithelial Cells

Current pharmacotherapies for symptomatic benign prostatic hyperplasia (BPH), an androgen receptor-driven, inflammatory disorder affecting elderly men, include 5α-reductase (5AR) inhibitors (i.e. dutasteride and finasteride) to block the conversion of testosterone to the more potent androgen receptor ligand dihydrotestosterone. Because dihydrotestosterone is the precursor for estrogen receptor β (ERβ) ligands, 5AR inhibitors could potentially limit ERβ activation, which maintains prostate tissue homeostasis. We have uncovered signaling pathways in BPH-derived prostate epithelial cells (BPH-1) that are impacted by 5AR inhibition. The induction of apoptosis and repression of the cell adhesion protein E-cadherin by the 5AR inhibitor dutasteride requires both ERβ and TGFβ. Dutasteride also induces cyclooxygenase type 2 (COX-2), which functions in a negative feedback loop in TGFβ and ERβ signaling pathways as evidenced by the potentiation of apoptosis induced by dutasteride or finasteride upon pharmacological inhibition or shRNA-mediated ablation of COX-2. Concurrently, COX-2 positively impacts ERβ action through its effect on the expression of a number of steroidogenic enzymes in the ERβ ligand metabolic pathway. Therefore, effective combination pharmacotherapies, which have included non-steroidal anti-inflammatory drugs, must take into account biochemical pathways affected by 5AR inhibition and opposing effects of COX-2 on the tissue-protective action of ERβ.