DeFranco Lab

The DeFranco laboratory has spent over 30 years examining glucocorticoid receptor function focusing predominantly on the mechanisms of receptor transactivation, interaction with coactivators, subcellular and subnuclear trafficking, interactions with molecular chaperones and processing. Over the years they have utilized various experimental model systems to provide novel mechanistic insights into receptor action. Some of their studies utilized neuronal cell systems and in particular using cells of hippocampal origin we provided one of the first demonstrations of developmentally regulated receptor degradation that is likely to be influenced by a receptor co-chaperone (e.g. CHIP). In the past few years they have initiated collaborations with Dr. Paula Monaghan-Nichols, a developmental neurobiologist, Dr. Selma Witchel, a pediatric endocrinologist and Dr. Anthony Rudine, a neonatologist, to expand our analysis of glucocorticoid receptor function to an area of clinical relevance.  Clinical and preclinical studies of postnatal and antenatal glucocorticoid administration suggest that detrimental effects of these hormones on neural function in adults and juveniles may be caused by alterations in the proliferation and differentiation of embryonic neural stem cells. The DeFranco laboratory therefore utilizes state-of-the-art genomic approaches, unique transgenic models and primary neural stem/progenitor cell cultures to uncover molecular mechanisms responsible for the detrimental effects of glucocorticoids on the developing brain.
 
The DeFranco laboratory also studies androgen receptor and estrogen receptor function with a particular emphasis on examining coregulators that  impact androgen action in prostate and the regulation of estrogen receptor function by oxidative stress and TGFß signaling. For example, they have identified a novel class of androgen receptor coregulators that are members of the Group III LIM domain family that includes the focal adhesion protein paxillin. Hic-5 is one such group III LIM domain protein highly related to paxillin that they have characterized as an androgen receptor and vitamin D receptor coregulator. Interestingly Hic-5 expression is restricted to the stromal compartment of the prostate gland and represents a novel tissue-specific coregulator that influences stromal/epithelial communication mediated by androgens and vitamin D. Their most recent work has identified a novel paracrine communication network between prostate epithelial and stromal cells that impacts estrogen receptor beta function.
 

Below displays the Research Details from the profile of each member of the lab.

Donald B. DeFranco, PhD

Golgi impregnation stain showing reduced complexity of pyramidal neurons in 10-day old mouse hippocampus (CA1 region) following in utero exposure to vehicle (Veh) or the dexamethasone (Dex).

Glucocorticoid Action in the Developing Brain

The life threatening, emotional and economic burdens of premature birth (~12% of pregnancies) have been greatly alleviated by antenatal treatment with synthetic glucocorticoids (sGCs). Antenatal sGCs accelerate tissue development reducing respiratory distress syndrome and intraventricular hemorrhage in premature infants, but they can affect developmental processes in the brain and trigger adverse behavioral and metabolic outcomes later in life. While the well-established beneficial neonatal outcomes of antenatal GCs support their continued use, we are utilizing mechanistic analysis of novel GC pathways in the developing brain (particularly in the hypothalamus and cerebral cortex) to identify new biomarkers and drug targets for male and/or female fetuses at high risk for adverse, neurological outcomes of GCs or resistant to their beneficial effects on the developing cerebral vasculature. Our approach includes molecular studies, genome wide assessment of sGC target genes, behavioral analyses in mice and histological analysis in knock-in mouse models as well as primary human neural stem cell cultures. Of direct clinical relevance are our proposed studies with cells derived from human fetuses that were exposed to antenatal GCs, which could identify novel real time biomarkers that predict outcome in children exposed to sGCs in utero.

Indirect immunofluorescence staining of murine embryonic neural stem cell cultures differentiated in vitro to visualize the stem cell marker nestin (green), the oligodendrocyte progenitor marker Olig2 (pink) and nuclei (DAPI in blue). Biological effects of GCs are mediated by the glucocorticoid receptor (GR), a member of the nuclear receptor super-family of transcription factors. While primarily studied for its role in the nucleus, GR is also localized in the plasma membrane where it can rapidly mobilize a variety of cytoplasmic signaling pathways. We have identified unique genomic (i.e. nuclear) and nongenomic (i.e. cytoplasmic) GR signaling pathways that are activated in response to antenatal GCs in the developing brain and cultured embryonic neural stem cells. For example, novel gender specific molecular targets of the genomic GR pathway are being identified by genome-wide RNA-Seq and ChiP-Seq approaches while work continues to investigate the biological impact of nongenomic GC effects on gap junction communication and synchronous, spontaneous Ca2+ transients in coupled neural stem cells. Furthermore, we continue to explore the impact of crosstalk between nongenomic and genomic GC pathways, which we have found to regulate site-specific GR phosphorylation and recruitment of the receptor to distinct subsets of target genes.


Estrogen Action in Benign and Malignant Prostatic Disease

Prostatic inflammation is a common feature of symptomatic benign prostatic hyperplasia (BPH) and may alter epithelial cell proliferation and tissue homeostasis in BPH through cytokine induction of proinflammatory signaling mediators such as cyclooxygenase-2 (Cox-2). Current therapies for symptomatic benign prostatic hyperplasia (BPH), an androgen receptor (AR) 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 AR ligand dihydrotestosterone (DHT). Since DHT 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 influenced 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 Transforming growth factor-beta (TGF-β). Dutasteride also induces COX-2 expression, 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 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.

Indirect immunofluorescence staining of E-cadherin (red) in 3-dimensional cultures of human prostate epithelial cells (DAPI visualized nuclei in blue). Our laboratory seeks to identify novel gene signatures that can be utilized as biomarkers for symptomatic BPH patients to predict response to NSAIDs or to direct therapeutic development towards a drugable target in BPH, ERß. We utilize 3D cultures of human prostate epithelial cells (PrECs) to provide mechanistic insights into the putative loss of epithelial integrity in BPH. Finally, the novel, unique genetic changes (i.e. gene signatures) associated with inflammatory mediators (i.e. Cox-2) or responsive to NSAIDs in cultured human PrECs are being assessed in patient samples, including from a clinical trial that utilizes combined 5AR and NSAID treatment. Thus, these studies will directly test the utility of identified gene signatures for predicting BPH patient responses to NSAIDs and stimulate the development of new drugs that target ERß and/or enhance the therapeutic efficacy of NSAIDs.


Krystle Frahm, PhD

Dr. Frahm's research interests focus on how endogenous and exogenous factors disrupt neuroendocrine development and function resulting in long-lasting physiological and behavioral consequences. Current studies are examining the role of glucocorticoid signaling during fetal development and in adulthood using genome-wide analysis and behavioral phenotyping. These collectively will provide insights into how altered hypothalamic output and can contribute to altered energy homeostasis and stress responses in a sex-specific manner.

Donald B. DeFranco, PhD
Professor & Vice Chair for Medical Education, Associate Dean for Medical Student Research


Krystle Frahm, PhD
Research Assistant Professor

Donald B. DeFranco, PhD

Journal Articles

Liu TT, Grubisha MJ, Frahm KA, Wendell SG, Liu J, Ricke WA, Auchus RJ & DeFranco DB (2016). Opposing effects of cyclooxygenase-2 on estrogen receptor-ß response to 5a-reductase inhibition in prostate epithelial cells. J. Biol. Chem. 291:14747-14760.  PMCID:  PMC4938192

 
Frahm KA, Peffer ME, Zhang JY, Luthra S, Chakka AB, Couger MB, Chandran UR, Monaghan AP & DeFranco DB (2016). The dexamethasone transcriptome in hypothalamic embryonic neural stem cells. Molecular Endocrin. 30, 144-154. PMCID:  PMC4695633
 
Leach DA, Need EF, Toivanen R, Trotta AP, Palenthorpe HM, Tamblyn DJ, Kopsaftis T, England GM, Smith E, Drew PA, Pinnock CB, Lee P, Holst J, Risbridger GP, Chopra S, DeFranco DB, Taylor RA and Buchanan G.  Stromal androgen receptor regulates the composition of the microenvironment to influence prostate cancer outcome.  Oncotarget 6:16135-16150, 2015.

Solomon JD, Heitzer MD, Liu TT, Beumer JH, Parise RA, Normolle DP, Leach DA, Buchanan G and DeFranco DB.  VDR activity is differentially affected by Hic-5 in prostate cancer and stromal cells. Mol Cancer Res 12:1166-1180, 2014.

Peffer ME, Chandran UR, Luthra S, Volonte D, Galbiati F, Garabedian MJ, Monaghan AP and DeFranco DB.  Caveolin-1 regulates genomic action of the glucocorticoid receptor in neural stem cells.  Mol Cell Biol 34:2611-2623, 2014.

Indyk JA, Candido-Vitto C, Wolf IM, Venkataraman S, Munoz R, Saladino RA, Witchel SF and DeFranco DB.  Reduced glucocorticoid receptor protein expression in children with critical illness.  Horm Res Paediatr 79:169-178, 2013.

Samarasinghe RA, Di Maio R, Volonte D, Galbiati F, Lewis M, Romero G and DeFranco DB. Nongenomic glucocorticoid receptor action regulates gap junction intercellular communication and neural progenitor cell proliferation. Proc Natl Acad Sci USA 108:16657-16662, 2011.


Krystle Frahm, PhD

Journal Articles

Frahm KA, Waldman JK, Luthra S, Rudine AC, Monaghan-Nichols AP, Chandran U and, DeFranco DB. A comparison of the sexually dimorphic dexamethasone transcriptome in mouse cerebral cortical and hypothalamic embryonic neural stem cells. Mol Cell Endocrinol 471:42-50,2018.
Frahm KA, Handa RJ and Tobet SA. Embryonic exposure to dexamethasone impacts non-neuronal cells in the adult paraventricular nucleus of the hypothalamus. J Endocr Soc 2(2):140-153, 2017.
Liu TT, Grubisha MJ, Frahm KA, Wendell SG, Liu J, Ricke WA, Auchus RJ and DeFranco DB. (2016). Opposing effects of COX-2 on ER-beta response to 5alpha-reductase inhibition in prostate epithelial cells. J Biol Chem 291(28):14747-14760, 2016.
Frahm KA, Peffer ME, Zhang JY, Luthra S, Chakka AB, Couger MB, Chandran UR, Monaghan-Nichols AP and  DeFranco DB. The dexamethasone transcriptome in hypothalamic embryonic neural stem cells. Mol Endocrinol 30(1):144-154, 2016.
Frahm KA and Tobet SA. Development of the blood-brain barrier within the paraventricular nucleus of the hypothalamus: Influence of fetal glucocorticoid excess. Brain Struct Funct 220(4):2225-2234, 2015.
Frahm KA, Nash CP and Tobet SA. Endocan immunoreactivity in the mouse brain: Method for identifying nonfunctional blood vessels. J Immunol Methods 398-399:27-32, 2013. 
Frahm KA, Schow MJ and Tobet SA. The vasculature within the paraventricular nucleus of the hypothalamus in mice varies as a function of development, sub-nuclear location, and GABA signaling. Horm Metab Res 44(8):619-624, 2012.