FKBP51 and the molecular chaperoning of metabolism.

The molecular chaperone FK506-binding protein 51 (FKBP51) is gaining attention as a meaningful biomarker of metabolic dysfunction. This review examines the emerging contributions of FKBP51 in adipogenesis and lipid metabolism, myogenesis and protein catabolism, and glucocorticoid-induced skin hypoplasia and dermal adipocytes. The FKBP51 signaling mechanisms that may explain these metabolic consequences are discussed. These mechanisms are diverse, with FKBP51 independently and directly regulating phosphorylation cascades and nuclear receptors. We provide a discussion of the newly developed compounds that antagonize FKBP51, which may offer therapeutic advantages for adiposity. These observations suggest we are only beginning to uncover the complex nature of FKBP51 and its molecular chaperoning of metabolism.

COUP-TFII revisited: Its role in metabolic gene regulation.

Chicken Ovalbumin Upstream Promoter Transcription Factor II (COUP-TFII) is an orphan member of the nuclear receptor family of transcriptional regulators. Although hormonal activation of COUP-TFII has not yet been identified, rodent genetic models have uncovered vital and diverse roles for COUP-TFII in biological processes. These include control of cardiac function and angiogenesis, reproduction, neuronal development, cell fate and organogenesis. Recently, an emerging body of evidence has demonstrated COUP-TFII involvement in various metabolic systems such as adipogenesis, lipid metabolism, hepatic gluconeogenesis, insulin secretion, and regulation of blood pressure. The potential relevance of these observations to human pathology has been corroborated by the identification of single nucleotide polymorphism in the human COUP-TFII promoter controlling insulin sensitivity. Of particular interest to metabolism is the ability of COUP-TFII to interact with the Glucocorticoid Receptor (GR). This interaction is known to control gluconeogenesis, principally through direct binding of COUP-TFII/GR complexes to the promoters of gluconeogenic enzyme genes. However, it is likely that this interaction is critical to other metabolic processes, since GR, like COUP-TFII, is an essential regulator of adipogenesis, insulin sensitivity, and blood pressure. This review will highlight these unique roles of COUP-TFII in metabolic gene regulation.

Deletion of the glucocorticoid receptor chaperone FKBP51 prevents glucocorticoid-induced skin atrophy.

FKBP51 (FK506-binding protein 51) is a known co-chaperone and regulator of the glucocorticoid receptor (GR), which usually attenuates its activity. FKBP51 is one of the major GR target genes in skin, but its role in clinical effects of glucocorticoids is not known. Here, we used FKBP51 knockout (KO) mice to determine FKBP51's role in the major adverse effect of topical glucocorticoids, skin atrophy. Unexpectedly, we found that all skin compartments (epidermis, dermis, dermal adipose and CD34+ stem cells) in FKBP51 KO animals were much more resistant to glucocorticoid-induced hypoplasia. Furthermore, despite the absence of inhibitory FKBP51, the basal level of expression and glucocorticoid activation of GR target genes were not increased in FKBP51 KO skin or CRISPR/Cas9-edited FKBP51 KO HaCaT human keratinocytes. FKBP51 is known to negatively regulate Akt and mTOR. We found a significant increase in AktSer473 and mTORSer2448 phosphorylation and downstream pro-growth signaling in FKBP51-deficient keratinocytes in vivo and in vitro. As Akt/mTOR-GR crosstalk is usually negative in skin, our results suggest that Akt/mTOR activation could be responsible for the lack of increased GR function and resistance of FKBP51 KO mice to the steroid-induced skin atrophy.

Protein phosphatase 5 and the tumor suppressor p53 down-regulate each other's activities in mice.

Protein phosphatase 5 (PP5), a serine/threonine phosphatase, has a wide range of biological functions and exhibits elevated expression in tumor cells. We previously reported that pp5-deficient mice have altered ataxia-telangiectasia mutated (ATM)-mediated signaling and function. However, this regulation was likely indirect, as ATM is not a known PP5 substrate. In the current study, we found that pp5-deficient mice are hypersensitive to genotoxic stress. This hypersensitivity was associated with the marked up-regulation of the tumor suppressor tumor protein p53 and its downstream targets cyclin-dependent kinase inhibitor 1A (p21), MDM2 proto-oncogene (MDM2), and phosphatase and tensin homolog (PTEN) in pp5-deficient tissues and cells. These observations suggested that PP5 plays a role in regulating p53 stability and function. Experiments conducted with p53+/-pp5+/- or p53+/-pp5-/- mice revealed that complete loss of PP5 reduces tumorigenesis in the p53+/- mice. Biochemical analyses further revealed that PP5 directly interacts with and dephosphorylates p53 at multiple serine/threonine residues, resulting in inhibition of p53-mediated transcriptional activity. Interestingly, PP5 expression was significantly up-regulated in p53-deficient cells, and further analysis of pp5 promoter activity revealed that p53 strongly represses PP5 transcription. Our results suggest a reciprocal regulatory interplay between PP5 and p53, providing an important feedback mechanism for the cellular response to genotoxic stress.

Protein Phosphatase PP5 Controls Bone Mass and the Negative Effects of Rosiglitazone on Bone through Reciprocal Regulation of PPARγ (Peroxisome Proliferator-activated Receptor γ) and RUNX2 (Runt-related Transcription Factor 2).

Peroxisome proliferator-activated receptor γ (PPARγ) and runt-related transcription factor 2 (RUNX2) are key regulators of mesenchymal stem cell (MSC) differentiation toward adipocytes and osteoblasts, respectively. Post-translational modifications of these factors determine their activities. Dephosphorylation of PPARγ at Ser-112 is required for its adipocytic activity, whereas phosphorylation of RUNX2 at serine 319 (Ser-319) promotes its osteoblastic activity. Here we show that protein phosphatase 5 (PP5) reciprocally regulates each receptor by targeting each serine. Mice deficient in PP5 phosphatase have increased osteoblast numbers and high bone formation, which results in high bone mass in the appendicular and axial skeleton. This is associated with a substantial decrease in lipid-containing marrow adipocytes. Indeed, in the absence of PP5 the MSC lineage allocation is skewed toward osteoblasts and away from lipid accumulating adipocytes, although an increase in beige adipocyte gene expression is observed. In the presence of rosiglitazone, PP5 translocates to the nucleus, binds to PPARγ and RUNX2, and dephosphorylates both factors, resulting in activation of PPARγ adipocytic and suppression of RUNX2 osteoblastic activities. Moreover, shRNA knockdown of PP5 results in cells refractory to rosiglitazone treatment. Lastly, mice deficient in PP5 are resistant to the negative effects of rosiglitazone on bone, which in wild type animals causes a 50% decrease in trabecular bone mass. In conclusion, PP5 is a unique phosphatase reciprocally regulating PPARγ and RUNX2 activities in marrow MSC.

FKBP51 Null Mice Are Resistant to Diet-Induced Obesity and the PPARγ Agonist Rosiglitazone.

FK506-binding protein-51 (FKBP51) is a molecular cochaperone recently shown to be a positive regulator of peroxisome proliferator-activated receptor (PPAR)γ, the master regulator of adipocyte differentiation and function. In cellular models of adipogenesis, loss of FKBP51 not only reduced PPARγ activity but also reduced lipid accumulation, suggesting that FKBP51 knock-out (KO) mice might have insufficient development of adipose tissue and lipid storage ability. This model was tested by examining wild-type (WT) and FKBP51-KO mice under regular and high-fat diet conditions. Under both diets, FKBP51-KO mice were resistant to weight gain, hepatic steatosis, and had greatly reduced white adipose tissue (WAT) but higher amounts of brown adipose tissue. Under high-fat diet, KO mice were highly resistant to adiposity and exhibited reduced plasma lipids and elevated glucose and insulin tolerance. Profiling of perigonadal and sc WAT revealed elevated expression of brown adipose tissue lineage genes in KO mice that correlated increased energy expenditure and a shift of substrate oxidation to carbohydrates, as measured by indirect calorimetry. To directly test PPARγ involvement, WT and KO mice were fed rosiglitazone agonist. In WT mice, rosiglitazone induced whole-body weight gain, increased WAT mass, a shift of substrate oxidation to lipids, and elevated expression of PPARγ-regulated lipogenic genes in WAT. In contrast, KO mice had reduced rosiglitazone responses for these parameters. Our results identify FKBP51 as an important regulator of PPARγ in WAT and as a potential new target in the treatment of obesity and diabetes.

Timcodar (VX-853) Is a Non-FKBP12 Binding Macrolide Derivative That Inhibits PPARγ and Suppresses Adipogenesis.

Nutrient overload and genetic factors have led to a worldwide epidemic of obesity that is the underlying cause of diabetes, atherosclerosis, and cardiovascular disease. In this study, we used macrolide drugs such as FK506, rapamycin, and macrolide derived, timcodar (VX-853), to determine their effects on lipid accumulation during adipogenesis. Rapamycin and FK506 bind to FK506-binding proteins (FKBPs), such as FKBP12, which causes suppression of the immune system and inhibition of mTOR. Rapamycin has been previously reported to inhibit the adipogenic process and lipid accumulation. However, rapamycin treatment in rodents caused immune suppression and glucose resistance, even though the mice lost weight. Here we show that timcodar (1 µM), a non-FKBP12-binding drug, significantly (p < 0.001) inhibited lipid accumulation during adipogenesis. A comparison of the same concentration of timcodar (1 µM) and rapamycin (1 µM) showed that both are inhibitors of lipid accumulation during adipogenesis. Importantly, timcodar potently (p < 0.01) suppressed transcriptional regulators of adipogenesis, PPARγ and C/EBPα, resulting in the inhibition of genes involved in lipid accumulation. These studies set the stage for timcodar as a possible antiobesity therapy, which is rapidly emerging as a pandemic.

PPARα (Peroxisome Proliferator-activated Receptor α) Activation Reduces Hepatic CEACAM1 Protein Expression to Regulate Fatty Acid Oxidation during Fasting-refeeding Transition.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is expressed at high levels in the hepatocyte, consistent with its role in promoting insulin clearance in liver. CEACAM1 also mediates a negative acute effect of insulin on fatty acid synthase activity. Western blot analysis reveals lower hepatic CEACAM1 expression during fasting. Treating of rat hepatoma FAO cells with Wy14,643, an agonist of peroxisome proliferator-activated receptor α (PPARα), rapidly reduces Ceacam1 mRNA and CEACAM1 protein levels within 1 and 2 h, respectively. Luciferase reporter assay shows a decrease in the promoter activity of both rat and mouse genes by Pparα activation, and 5'-deletion and block substitution analyses reveal that the Pparα response element between nucleotides -557 and -543 is required for regulation of the mouse promoter activity. Chromatin immunoprecipitation analysis demonstrates binding of liganded Pparα toCeacam1promoter in liver lysates ofPparα(+/+), but notPparα(-/-)mice fed a Wy14,643-supplemented chow diet. Consequently, Wy14,643 feeding reduces hepatic Ceacam1 mRNA and CEACAM1 protein levels, thus decreasing insulin clearance to compensate for compromised insulin secretion and maintain glucose homeostasis and insulin sensitivity in wild-type mice. Together, the data show that the low hepatic CEACAM1 expression at fasting is mediated by Pparα-dependent mechanisms. Changes in CEACAM1 expression contribute to the coordination of fatty acid oxidation and insulin action in the fasting-refeeding transition.

The glucocorticoid receptor: cause of or cure for obesity?

Glucocorticoid hormones (GCs) are important regulators of lipid metabolism, promoting lipolysis with acute treatment but lipogenesis with chronic exposure. Conventional wisdom posits that these disparate outcomes are mediated by the classical glucocorticoid receptor GRα. There is insufficient knowledge of the GC receptors (GRα and GRß) in metabolic conditions such as obesity and diabetes. We present acute models of GC exposure that induce lipolysis, such as exercise, as well as chronic-excess models that cause obesity and lipid accumulation in the liver, such as hepatic steatosis. Alternative mechanisms are then proposed for the lipogenic actions of GCs, including induction of GC resistance by the GRß isoform, and promotion of lipogenesis by GC activation of the mineralocorticoid receptor (MR). Finally, the potential involvement of chaperone proteins in the regulation of adipogenesis is considered. This reevaluation may prove useful to future studies on the steroidal basis of adipogenesis and obesity.

Analysis of FK506, timcodar (VX-853) and FKBP51 and FKBP52 chaperones in control of glucocorticoid receptor activity and phosphorylation.

The immunosuppressive ligand FK506 and the FK506-binding protein FKBP52 are stimulatory to glucocorticoid receptor (GR) activity. Here, we explore the underlying mechanism by comparing GR activity and phosphorylation status in response to FK506 and the novel nonimmunosuppressive ligand timcodar (VX-853) and in the presence and absence of FKBP52 and the closely related protein FKBP51. Using mouse embryonic fibroblast cells (MEFs) deficient knockout (KO) in FKBP51 or FKBP52, we show decreased GR activity at endogenous genes in 52KO cells, but increased activity in 51KO cells. In 52KO cells, elevated phosphorylation occurred at inhibitory serine 212 and decreased phosphorylation at the stimulatory S220 residue. In contrast, 51KO cells showed increased GR phosphorylation at the stimulatory residues S220 and S234. In wild-type (WT) MEF cells, timcodar, like FK506, potentiated dexamethasone-induced GR transcriptional activity at two endogenous genes. Using 52KO and 51KO MEF cells, FK506 potentiated GR activity in 51KO cells but could not do so in 52KO cells, suggesting FKBP52 as the major target of FK506 action. Like FK506, timcodar potentiated GR in 51KO cells, but it also increased GR activity in 52KO cells. Knock-down of FKBP51 in the 52KO cells showed that the latter effect of timcodar required FKBP51. Thus, timcodar appears to have a dual specificity for FKBP51 and FKBP52. This work demonstrates phosphorylation as an important mechanism in FKBP control of GR and identifies the first nonimmunosuppressive macrolide capable of targeting GR action.

FKBP51 controls cellular adipogenesis through p38 kinase-mediated phosphorylation of GRα and PPARγ.

Glucocorticoid receptor-α (GRα) and peroxisome proliferator-activated receptor-γ (PPARγ) are critical regulators of adipogenic responses. We have shown that FK506-binding protein 51 (FKBP51) represses the Akt-p38 kinase pathway to reciprocally inhibit GRα but stimulate PPARγ by targeting serine 112 (PPARγ) and serines 220 and 234 (GRα). Here, this mechanism is shown to be essential for GRα and PPARγ control of cellular adipogenesis. In 3T3-L1 cells, FKBP51 was a prominent marker of the differentiated state and knockdown of FKBP51 showed reduced lipid accumulation and expression of adipogenic genes. Compared with wild-type (WT), FKBP51 knockout (51KO) mouse embryonic fibroblasts (MEFs) showed dramatic resistance to differentiation, with almost no lipid accumulation and greatly reduced adipogenic gene expression. These features were rescued by reexpression of FKBP51 in 51KO cells. 51KO MEFs exhibited reduced fatty acid synthase activity, increased sensitivity to GRα-induced lipolysis, and reduced PPARγ activity at adipogenic genes (adiponectin, CD36, and perilipin) but elevated GRα transrepression at these same genes. A p38 kinase inhibitor increased lipid content in WT cells and also restored lipid levels in 51KO cells, showing that elevated p38 kinase activity is a major contributor to adipogenic resistance in the 51KO cells. In 51KO cells, the S112A mutant of PPARγ and the triple S212A/S220A/S234A mutant of GRα both increased lipid accumulation, identifying these residues as targets of the FKBP51/p38 axis. Our combined investigations have uncovered FKBP51 as a key regulator of adipogenesis via the Akt-p38 pathway and as a potential target in the treatment of obesity and related disorders.

FKBP51 reciprocally regulates GRα and PPARγ activation via the Akt-p38 pathway.

FK506-binding protein 51 (FKBP51) is a negative regulator of glucocorticoid receptor-α (GRα), although the mechanism is unknown. We show here that FKBP51 is also a chaperone to peroxisome proliferator-activated receptor-γ (PPARγ), which is essential for activity, and uncover the mechanism underlying this differential regulation. In COS-7 cells, FKBP51 overexpression reduced GRα activity at a glucocorticoid response element-luciferase reporter, while increasing PPARγ activity at a peroxisome proliferator response element reporter. Conversely, FKBP51-deficient (knockout) (51KO) mouse embryonic fibroblasts (MEFs) showed elevated GRα but reduced PPARγ activities compared with those in wild-type MEFs. Phosphorylation is known to exert a similar pattern of reciprocal modulation of GRα and PPARγ. Knockdown of FKBP51 in 3T3-L1 preadipocytes increased phosphorylation of PPARγ at serine 112, a phospho-residue that inhibits activity. In 51KO cells, elevated phosphorylation of GRα at serines 220 and 234, phospho-residues that promote activity, was observed. Because FKBP51 is an essential chaperone to the Akt-specific phosphatase PH domain leucine-rich repeat protein phosphatase, Akt signaling was investigated. Elevated Akt activation and increased activation of p38 kinase, a downstream target of Akt that phosphorylates GRα and PPARγ, were seen in 51KO MEFs, causing activation and inhibition, respectively. Inactivation of p38 with PD169316 reversed the effects of FKBP51 deficiency on GRα and PPARγ activities and reduced PPARγ phosphorylation. Last, loss of FKBP51 caused a shift of PPARγ from cytoplasm to nucleus, as previously shown for GRα. A model is proposed in which FKBP51 loss reciprocally regulates GRα and PPARγ via 2 complementary mechanisms: activation of Akt-p38-mediated phosphorylation and redistribution of the receptors to the nucleus for direct targeting by p38.

Suppression of protein kinase C theta contributes to enhanced myogenesis in vitro via IRS1 and ERK1/2 phosphorylation.

BACKGROUND: Differentiation and fusion of skeletal muscle myoblasts into multi-nucleated myotubes is required for neonatal development and regeneration in adult skeletal muscle. Herein, we report novel findings that protein kinase C theta (PKCθ) regulates myoblast differentiation via phosphorylation of insulin receptor substrate-1 and ERK1/2. RESULTS: In this study, PKCθ knockdown (PKCθshRNA) myotubes had reduced inhibitory insulin receptor substrate-1 ser1095 phosphorylation, enhanced myoblast differentiation and cell fusion, and increased rates of protein synthesis as determined by [3H] phenylalanine incorporation. Phosphorylation of insulin receptor substrate-1 ser632/635 and extracellular signal-regulated kinase1/2 (ERK1/2) was increased in PKCθshRNA cells, with no change in ERK5 phosphorylation, highlighting a PKCθ-regulated myogenic pathway. Inhibition of PI3-kinase prevented cell differentiation and fusion in control cells, which was attenuated in PKCθshRNA cells. Thus, with reduced PKCθ, differentiation and fusion occur in the absence of PI3-kinase activity. Inhibition of the ERK kinase, MEK1/2, impaired differentiation and cell fusion in control cells. Differentiation was preserved in PKCθshRNA cells treated with a MEK1/2 inhibitor, although cell fusion was blunted, indicating PKCθ regulates differentiation via IRS1 and ERK1/2, and this occurs independently of MEK1/2 activation. CONCLUSION: Cellular signaling regulating the myogenic program and protein synthesis are complex and intertwined. These studies suggest that PKCθ regulates myogenic and protein synthetic signaling via the modulation of IRS1and ERK1/2 phosphorylation. Myotubes lacking PKCθ had increased rates of protein synthesis and enhanced myotube development despite reduced activation of the canonical anabolic-signaling pathway. Further investigation of PKCθ regulated signaling may reveal important interactions regulating skeletal muscle health in an insulin resistant state.

Chaperoning steroidal physiology: lessons from mouse genetic models of Hsp90 and its cochaperones.

The molecular chaperone Hsp90 is abundant, ubiquitous, and catholic to biological processes in eukaryotes, controlling phosphorylation cascades, protein stability and turnover, client localization and trafficking, and ligand-receptor interactions. Not surprisingly, Hsp90 does not accomplish these activities alone. Instead, an ever-growing number of cochaperones have been identified, leading to an explosion of reports on their molecular and cellular effects on Hsp90 chaperoning of client substrates. Notable among these clients are many members of the steroid receptor family, such as glucocorticoid, androgen, estrogen and progesterone receptors. Cochaperones typically associated with the mature, hormone-competent states of these receptors include p23, the FK506-binding protein 52 (FKBP52), FKBP51, protein phosphatase 5 (PP5) and cyclophilin 40 (Cyp40). The ultimate relevance of these cochaperones to steroid receptor action depends on their physiological effects. In recent years, the first mouse genetic models of these cochaperones have been developed. This work will review the complex and intriguing phenotypes so far obtained in genetically-altered mice and compare them to the known molecular and cellular impacts of cochaperones on steroid receptors. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).

FKBP38 peptidylprolyl isomerase promotes the folding of cystic fibrosis transmembrane conductance regulator in the endoplasmic reticulum.

FK506-binding protein 38 (FKBP38), a membrane-anchored, tetratricopeptide repeat (TPR)-containing immunophilin, associates with nascent plasma membrane ion channels in the endoplasmic reticulum (ER). It promotes the maturation of the human ether-à-go-go-related gene (HERG) potassium channel and maintains the steady state level of the cystic fibrosis transmembrane conductance regulator (CFTR), but the underlying mechanisms remain unclear. Using a combination of steady state and pulse-chase analyses, we show that FKBP38 knockdown increases protein synthesis but inhibits the post-translational folding of CFTR, leading to reduced steady state levels of CFTR in the ER, decreased processing, and impaired cell surface functional expression in Calu-3 human airway epithelial cells. The membrane anchorage of FKBP38 is necessary for the inhibition of protein synthesis but not for CFTR post-translational folding. In contrast, the peptidylprolyl cis/trans isomerase active site is utilized to promote CFTR post-translational folding but is not important for regulation of protein synthesis. Uncoupling FKBP38 from Hsp90 by substituting a conserved lysine in the TPR domain modestly enhances CFTR maturation and further reduces its synthesis. Removing the N-terminal glutamate-rich domain (ERD) slightly enhances CFTR synthesis but reduces its maturation, suggesting that the ERD contributes to FKBP38 biological activities. Our data support a dual role for FKBP38 in regulating CFTR synthesis and post-translational folding. In contrast to earlier prediction but consistent with in vitro enzymological studies, FKBP38 peptidylprolyl cis/trans isomerase plays an important role in membrane protein biogenesis on the cytoplasmic side of the ER membrane, whose activity is negatively regulated by Hsp90 through the TPR domain.

Protein phosphatase 5 mediates lipid metabolism through reciprocal control of glucocorticoid receptor and peroxisome proliferator-activated receptor-γ (PPARγ).

Glucocorticoid receptor-α (GRα) and peroxisome proliferator-activated receptor-γ (PPARγ) regulate adipogenesis by controlling the balance between lipolysis and lipogenesis. Here, we show that protein phosphatase 5 (PP5), a nuclear receptor co-chaperone, reciprocally modulates the lipometabolic activities of GRα and PPARγ. Wild-type and PP5-deficient (KO) mouse embryonic fibroblast cells were used to show binding of PP5 to both GRα and PPARγ. In response to adipogenic stimuli, PP5-KO mouse embryonic fibroblast cells showed almost no lipid accumulation with reduced expression of adipogenic markers (aP2, CD36, and perilipin) and low fatty-acid synthase enzymatic activity. This was completely reversed following reintroduction of PP5. Loss of PP5 increased phosphorylation of GRα at serines 212 and 234 and elevated dexamethasone-induced activity at prolipolytic genes. In contrast, PPARγ in PP5-KO cells was hyperphosphorylated at serine 112 but had reduced rosiglitazone-induced activity at lipogenic genes. Expression of the S112A mutant rescued PPARγ transcriptional activity and lipid accumulation in PP5-KO cells pointing to Ser-112 as an important residue of PP5 action. This work identifies PP5 as a fulcrum point in nuclear receptor control of the lipolysis/lipogenesis equilibrium and as a potential target in the treatment of obesity.

FKBP51-a selective modulator of glucocorticoid and androgen sensitivity.

FK506-binding protein 51 (FKBP51) is gaining increased recognition for its essential roles in cell biology. Originally discovered as a component of steroid receptor complexes, it is now known to regulate a diverse set of transcription factors, enzymes and structural proteins. Its cellular properties suggest numerous possible functions for FKBP51 in physiology, and the best clue to its potential importance may be the following: FKBP51 is a glucocorticoid-induced negative regulator of the glucocorticoid receptor. Thus, FKBP51 is intricately involved in regulation of the most pleiotropic hormone known to biology. In contrast to glucocorticoid receptor, FKBP51 is a positive regulator of the androgen receptor, suggesting that it functions as a reciprocal modulator of glucocorticoid-mediated and androgen-mediated physiology. In this work, we evaluate this hypothesis by examining recent cellular and physiological evidence.

Fkbp52 regulates androgen receptor transactivation activity and male urethra morphogenesis.

Hypospadias is a common birth defect in humans, yet its etiology and pattern of onset are largely unknown. Recent studies have shown that male mice with targeted ablation of FK506-binding protein-52 (Fkbp52) develop hypospadias, most likely due to actions of Fkbp52 as a molecular co-chaperone of the androgen receptor (AR). Here, we further dissect the developmental and molecular mechanisms that underlie hypospadias in Fkbp52-deficient mice. Scanning electron microscopy revealed a defect in the elevation of prepucial swelling that led to the onset of the ventral penile cleft. Interestingly, expression of Fkbp52 was highest in the ventral aspect of the developing penis that undergoes fusion of the urethral epithelium. Although in situ hybridization and immunohistochemical analyses suggested that Fkbp52 mutants had a normal urethral epithelium signaling center and epithelial differentiation, a reduced apoptotic cell index at ventral epithelial cells at the site of fusion and a defect of genital mesenchymal cell migration were observed. Supplementation of gestating females with excess testosterone partially rescued the hypospadic phenotype in Fkbp52 mutant males, showing that loss of Fkbp52 desensitizes AR to hormonal activation. Direct measurement of AR activity was performed in mouse embryonic fibroblast cells treated with dihydrotestosterone or synthetic agonist R1881. Reduced AR activity at genes controlling sexual dimorphism and cell growth was found in Fkbp52-deficient mouse embryonic fibroblast cells. However, chromatin immunoprecipitation analysis revealed normal occupancy of AR at gene promoters, suggesting that Fkbp52 exerts downstream effects on the transactivation function of AR. Taken together, our data show Fkbp52 to be an important molecular regulator in the androgen-mediated pathway of urethra morphogenesis.

Discovery of glucocorticoid receptor-beta in mice with a role in metabolism.

Glucocorticoid hormones control diverse physiological processes, including metabolism and immunity, by activating the major glucocorticoid receptor (GR) isoform, GRalpha. However, humans express an alternative isoform, human (h)GRbeta, that acts as an inhibitor of hGRalpha to produce a state of glucocorticoid resistance. Indeed, evidence exists that hGRbeta contributes to many diseases and resistance to glucocorticoid hormone therapy. However, rigorous testing of the GRbeta contribution has not been possible, because rodents, especially mice, are not thought to express the beta-isoform. Here, we report expression of GRbeta mRNA and protein in the mouse. The mGRbeta isoform arises from a distinct alternative splicing mechanism utilizing intron 8, rather than exon 9 as in humans. The splicing event produces a form of beta that is similar in structure and functionality to hGRbeta. Mouse (m)GRbeta has a degenerate C-terminal region that is the same size as hGRbeta. Using a variety of newly developed tools, such as a mGRbeta-specific antibody and constructs for overexpression and short hairpin RNA knockdown, we demonstrate that mGRbeta cannot bind dexamethasone agonist, is inhibitory of mGRalpha, and is up-regulated by inflammatory signals. These properties are the same as reported for hGRbeta. Additionally, novel data is presented that mGRbeta is involved in metabolism. When murine tissue culture cells are treated with insulin, no effect on mGRalpha expression was observed, but GRbeta was elevated. In mice subjected to fasting-refeeding, a large increase of GRbeta was seen in the liver, whereas mGRalpha was unchanged. This work uncovers the much-needed rodent model of GRbeta for investigations of physiology and disease.