AKAP13 Enhances CREB1 Activation by FSH in Granulosa Cells.

Granulosa cells (GCs) must respond appropriately to follicle-stimulating hormone (FSH) for proper follicle maturation. FSH activates protein kinase A (PKA) leading to phosphorylation of the cyclic AMP response element binding protein-1 (CREB1). We identified a unique A-kinase anchoring protein (AKAP13) containing a Rho guanine nucleotide exchange factor (RhoGEF) region that was induced in GCs during folliculogenesis. AKAPs are known to coordinate signaling cascades, and we sought to evaluate the role of AKAP13 in GCs in response to FSH. Aromatase reporter activity was increased in COV434 human GCs overexpressing AKAP13. Addition of FSH, or the PKA activator forskolin, significantly enhanced this activity by 1.5- to 2.5-fold, respectively (p < 0.001). Treatment with the PKA inhibitor H89 significantly reduced AKAP13-dependent activation of an aromatase reporter (p = 0.0067). AKAP13 physically interacted with CREB1 in co-immunoprecipitation experiments and increased the phosphorylation of CREB1. CREB1 phosphorylation increased after FSH treatment in a time-specific manner, and this effect was reduced by siRNA directed against AKAP13 (p = 0.05). CREB1 activation increased by 18.5-fold with co-expression of AKAP13 in the presence of FSH (p < 0.001). Aromatase reporter activity was reduced by inhibitors of the RhoGEF region, C3 transferase and A13, and greatly enhanced by the RhoGEF activator, A02. In primary murine and COV43 GCs, siRNA knockdown of Akap13/AKAP13 decreased aromatase and luteinizing hormone receptor transcripts in cells treated with FSH, compared with controls. Collectively, these findings suggest that AKAP13 may function as a scaffolding protein in FSH signal transduction via an interaction with CREB, resulting in phosphorylation of CREB.

A-kinase anchoring protein 13 interacts with the vitamin D receptor to alter vitamin D-dependent gene activation in uterine leiomyoma cells.

OBJECTIVE: To determine if A-kinase anchoring protein 13 (AKAP13) interacts with the vitamin D receptor (VDR) to alter vitamin D-dependent signaling in fibroid cells. Uterine leiomyomas (fibroids) are characterized by a fibrotic extracellular matrix and are associated with vitamin D deficiency. Treatment with vitamin D (1,25-dihydroxyvitamin D3) reduces fibroid growth and extracellular matrix gene expression. A-kinase anchoring protein 13 is overexpressed in fibroids and interacts with nuclear hormone receptors, but it is not known whether AKAP13 may interact with the VDR to affect vitamin D signaling in fibroids. DESIGN: Laboratory studies. SETTING: Translational science laboratory. INTERVENTION(S): Human immortalized fibroid or myometrial cells were treated with 1,25-hydroxyvitamin D3 (1,25(OH)2D3) and transfected using expression constructs for AKAP13 or AKAP13 mutants, RhoQL, C3 transferase, or small interfering ribonucleic acids (RNAs). MAIN OUTCOME MEASURE(S): Messenger ribonucleic acid (mRNA) levels of AKAP13, fibromodulin, and versican as measured by quantitative real-time polymerase chain reaction. Glutathione S-transferase-binding assays. Vitamin D-dependent gene activation as measured by luciferase assays. RESULT(S): 1,25(OH)2D3 resulted in a significant reduction in mRNA levels encoding AKAP13, versican, and fibromodulin. Small interfering RNA silencing of AKAP13 decreased both fibromodulin and versican mRNA levels. Glutathione S-transferase-binding assays revealed that AKAP13 bound to the VDR through its nuclear receptor interacting region. Cotransfection of AKAP13 and VDR significantly reduced vitamin D-dependent gene activation. RhoA pathway inhibition partially relieved repression of vitamin D-dependent gene activation by AKAP13. CONCLUSION(S): These data suggest that AKAP13 inhibited the vitamin D receptor activation by a mechanism that required, at least in part, RhoA activation.

A-Kinase Anchoring Protein 13 (AKAP13) Augments Progesterone Signaling in Uterine Fibroid Cells.

Context: Uterine leiomyomata (fibroids) are prevalent sex hormone‒dependent tumors with an altered response to mechanical stress. Ulipristal acetate, a selective progesterone receptor (PR) modulator, significantly reduces fibroid size in patients. However, PR signaling in fibroids and its relationship to mechanical signaling are incompletely understood. Objective: Our prior studies revealed that A-kinase anchoring protein 13 (AKAP13) was overexpressed in fibroids and contributed to altered mechanotransduction in fibroids. Because AKAP13 augmented nuclear receptor signaling in other tissues, we sought to determine whether AKAP13 might influence PR signaling in fibroids. Methods and Results: Fibroid samples from patients treated with ulipristal acetate or placebo were examined for AKAP13 expression by using immunohistochemistry. In immortalized uterine fibroid cell lines and COS-7 cells, we observed that AKAP13 increased ligand-dependent PR activation of luciferase reporters and endogenous progesterone-responsive genes for PR-B but not PR-A. Inhibition of ERK reduced activation of PR-dependent signaling by AKAP13, but inhibition of p38 MAPK had no effect. In addition, glutathione S-transferase‒binding assays revealed that AKAP13 was bound to PR-B through its carboxyl terminus. Conclusion: These data suggest an intersection of mechanical signaling and PR signaling involving AKAP13 through ERK. Further elucidation of the integration of mechanical and hormonal signaling pathways in fibroids may provide insight into fibroid development and suggest new therapeutic strategies for treatment.

Molecular characterization of the human microbiome from a reproductive perspective.

The process of reproduction inherently poses unique microbial challenges because it requires the transfer of gametes from one individual to the other, meanwhile preserving the integrity of the gametes and individuals from harmful microbes during the process. Advances in molecular biology techniques have expanded our understanding of the natural organisms living on and in our bodies, including those inhabiting the reproductive tract. Over the past two decades accumulating evidence has shown that the human microbiome is tightly related to health and disease states involving the different body systems, including the reproductive system. Here we introduce the science involved in the study of the human microbiome. We examine common methods currently used to characterize the human microbiome as an inseparable part of the reproductive system. Finally, we consider a few limitations, clinical implications, and the critical need for additional research in the field of human fertility.

The Rho guanine nucleotide exchange factor AKAP13 (BRX) is essential for cardiac development in mice.

A fundamental biologic principle is that diverse biologic signals are channeled through shared signaling cascades to regulate development. Large scaffold proteins that bind multiple proteins are capable of coordinating shared signaling pathways to provide specificity to activation of key developmental genes. Although much is known about transcription factors and target genes that regulate cardiomyocyte differentiation, less is known about scaffold proteins that couple signals at the cell surface to differentiation factors in developing heart cells. Here we show that AKAP13 (also known as Brx-1, AKAP-Lbc, and proto-Lbc), a unique protein kinase A-anchoring protein (AKAP) guanine nucleotide exchange region belonging to the Dbl family of oncogenes, is essential for cardiac development. Cardiomyocytes of Akap13-null mice had deficient sarcomere formation, and developing hearts were thin-walled and mice died at embryonic day 10.5-11.0. Disruption of Akap13 was accompanied by reduced expression of Mef2C. Consistent with a role of AKAP13 upstream of MEF2C, Akap13 siRNA led to a reduction in Mef2C mRNA, and overexpression of AKAP13 augmented MEF2C-dependent reporter activity. The results suggest that AKAP13 coordinates Galpha(12) and Rho signaling to an essential transcription program in developing cardiomyocytes.

Ski-interacting protein, a bifunctional nuclear receptor coregulator that interacts with N-CoR/SMRT and p300.

Ski-interacting protein (SKIP), a vitamin D receptor (VDR) coactivator, also functions as a repressor in Notch signalling in association with the corepressor SMRT. Here we show that SKIP bifunctionally modulates (activates or represses) Retinoid-X receptor (RXR)- and VDR-dependent gene transcription in a cell line-specific manner, with activation in CV-1 and repression in P19 cells. The coactivator function of SKIP in these cells appeared to correlate with the relative level and ratio of expression of N-CoR and p300, with greater SKIP activation in higher p300-expressing and lower N-CoR-expressing cell-lines. C-terminal deletion of SKIP (delta334-536 aa) was associated with strong activation in both CV-1 and P19 cells. The corepressors N-CoR and SMRT and the coregulator p300 interacted with SKIP through the same N-terminal region (1-200 aa). Overall these results suggest that transcriptional action of SKIP may depend on distinct functional domains and cell line-specific interactions with both corepressors and coactivators.

Estrogen action and cytoplasmic signaling pathways. Part II: the role of growth factors and phosphorylation in estrogen signaling.

In recent years, distinct signaling pathways involving specific complexes of cytoplasmic proteins have been shown to orchestrate estrogen action. These pathways might supplement or augment genomic effects of estrogen that are attributable to transcriptional activation by liganded receptor. Signals might be transduced through phosphorylation of the estrogen receptors (ERs), or indirectly through effects upon transcriptional coactivators or cell receptors. Estrogen signaling is coupled to growth factor signaling with feedback mechanisms directly impacting function of growth factor receptors. These signaling pathways regulate important physiological processes, such as cell growth and apoptosis. Here, we focus on cytoplasmic signaling pathways leading to activation of ERs.

Estrogen action and cytoplasmic signaling cascades. Part I: membrane-associated signaling complexes.

Remarkable progress in recent years has suggested that estrogen action in vivo is complex and often involves activation of cytoplasmic signaling cascades in addition to genomic actions mediated directly through estrogen receptors alpha and beta. Rather than a linear response mediated solely through estrogen-responsive DNA elements, in vivo estrogen might simultaneously activate distinct signaling cascades that function as networks to coordinate tissue responses to estrogen. This complex signaling system provides for exquisite control and plasticity of response to estrogen at the tissue level, and undoubtedly contributes to the remarkable tissue-specific responses to estrogens. In part I of this series, we summarize cytoplasmic signaling modules involving estrogen or estrogen receptors, with particular focus on recently described membrane-associated signaling complexes.