Endothelial cell expression of a STING gain-of-function mutation initiates pulmonary lymphocytic infiltration.

Patients afflicted with Stimulator of interferon gene (STING) gain-of-function mutations frequently present with debilitating interstitial lung disease (ILD) that is recapitulated in mice expressing the STINGV154M mutation (VM). Prior radiation chimera studies revealed an unexpected and critical role for non-hematopoietic cells in initiating ILD. To identify STING-expressing non-hematopoietic cell types required for the development of ILD, we use a conditional knockin (CKI) model and direct expression of the VM allele to hematopoietic cells, fibroblasts, epithelial cells, or endothelial cells. Only endothelial cell-targeted VM expression results in enhanced recruitment of immune cells to the lung associated with elevated chemokine expression and the formation of bronchus-associated lymphoid tissue, as seen in the parental VM strain. These findings reveal the importance of endothelial cells as instigators of STING-driven lung disease and suggest that therapeutic targeting of STING inhibitors to endothelial cells could potentially mitigate inflammation in the lungs of STING-associated vasculopathy with onset in infancy (SAVI) patients or patients afflicted with other ILD-related disorders.

MDM2 Regulation of HIF Signaling Causes Microvascular Dysfunction in Hypertrophic Cardiomyopathy.

BACKGROUND: Microvasculature dysfunction is a common finding in pathologic remodeling of the heart and is thought to play an important role in the pathogenesis of hypertrophic cardiomyopathy (HCM), a disease caused by sarcomere gene mutations. We hypothesized that microvascular dysfunction in HCM was secondary to abnormal microvascular growth and could occur independent of ventricular hypertrophy. METHODS: We used multimodality imaging methods to track the temporality of microvascular dysfunction in HCM mouse models harboring mutations in the sarcomere genes Mybpc3 (cardiac myosin binding protein C3) or Myh6 (myosin heavy chain 6). We performed complementary molecular methods to assess protein quantity, interactions, and post-translational modifications to identify mechanisms regulating this response. We manipulated select molecular pathways in vivo using both genetic and pharmacological methods to validate these mechanisms. RESULTS: We found that microvascular dysfunction in our HCM models occurred secondary to reduced myocardial capillary growth during the early postnatal time period and could occur before the onset of myocardial hypertrophy. We discovered that the E3 ubiquitin protein ligase MDM2 (murine double minute 2) dynamically regulates the protein stability of both HIF1α (hypoxia-inducible factor 1 alpha) and HIF2α (hypoxia-inducible factor 2 alpha)/EPAS1 (endothelial PAS domain protein 1) through canonical and noncanonical mechanisms. The resulting HIF imbalance leads to reduced proangiogenic gene expression during a key period of myocardial capillary growth. Reducing MDM2 protein levels by genetic or pharmacological methods normalized HIF protein levels and prevented the development of microvascular dysfunction in both HCM models. CONCLUSIONS: Our results show that sarcomere mutations induce cardiomyocyte MDM2 signaling during the earliest stages of disease, and this leads to long-term changes in the myocardial microenvironment.

Mice lacking γENaC palmitoylation sites maintain benzamil-sensitive Na+ transport despite reduced channel activity.

Epithelial Na+ channels (ENaCs) control extracellular fluid volume by facilitating Na+ absorption across transporting epithelia. In vitro studies showed that Cys-palmitoylation of the γENaC subunit is a major regulator of channel activity. We tested whether γ subunit palmitoylation sites are necessary for channel function in vivo by generating mice lacking the palmitoylated cysteines (γC33A,C41A) using CRISPR/Cas9 technology. ENaCs in dissected kidney tubules from γC33A,C41A mice had reduced open probability compared with wild-type (WT) littermates maintained on either standard or Na+-deficient diets. Male mutant mice also had higher aldosterone levels than WT littermates following Na+ restriction. However, γC33A,C41A mice did not have reduced amiloride-sensitive Na+ currents in the distal colon or benzamil-induced natriuresis compared to WT mice. We identified a second, larger conductance cation channel in the distal nephron with biophysical properties distinct from ENaC. The activity of this channel was higher in Na+-restricted γC33A,C41A versus WT mice and was blocked by benzamil, providing a possible compensatory mechanism for reduced prototypic ENaC function. We conclude that γ subunit palmitoylation sites are required for prototypic ENaC activity in vivo but are not necessary for amiloride/benzamil-sensitive Na+ transport in the distal nephron or colon.

Expression of a STING Gain-of-function Mutation in Endothelial Cells Initiates Lymphocytic Infiltration of the Lungs.

Patients afflicted with STING gain-of-function mutations frequently present with debilitating interstitial lung disease ( ILD ) that is recapitulated in mice expressing the STING V154M mutation ( VM ). Prior radiation chimera studies revealed an unexpected and critical role for non-hematopoietic cells in the initiation of ILD. To identify STING-expressing non-hematopoietic cell types relevant to ILD, we generated a conditional knock-in ( CKI ) model in which expression of the VM allele was directed to hematopoietic cells, fibroblasts, epithelial cells, or endothelial cells. Only endothelial cell-targeted expression of the mutant allele resulted in the recruitment of immune cells to the lung and the formation of bronchus-associated lymphoid tissue, as seen in the parental VM strain. These findings reveal the importance of endothelial cells as instigators of STING-driven lung disease and suggest that therapeutic targeting of STING inhibitors to endothelial cells could potentially mitigate inflammation in the lungs of SAVI patients or patients afflicted with other ILD-related disorders. Summary: Patients with STING gain-of-function (GOF) mutations develop life-threatening lung autoinflammation. In this study, Gao et al. utilize a mouse model of conditional STING GOF to demonstrate a role for endothelial STING GOF in initiating immune cell recruitment into lung tissues of SAVI mice.

Differentiating primary from secondary lung cancer with FDG PET/CT and extra-pulmonary tumor grade.

OBJECTIVE: Assess feasibility of differentiating primary from secondary lung cancer in patients with a solid solitary malignant pulmonary lesion (SMPL) and a previously resected extrapulmonary tumor. METHODS: Patients with pathology proven primary or secondary lung cancer from a solitary pulmonary lesion and known histopathology of extrapulmonary tumor were included. Patients with a small pulmonary lesion size, multiple malignant pulmonary nodules or an active infectious/inflammatory process were excluded. Extrapulmonary tumor grade was categorized as low, intermediate and high and was matched to FDG uptake intensity of SMPL, with FDG uptake range (SMPL/Liver SUVmax) of <0.9 for low, 0.91-1.99 for intermediate and >2.0 for high extrapulmonary tumor grade. RESULTS: Of 274 patients, 62 met the study criteria. 46 are primary and 16 are secondary lung cancer. There are 19 low, 27 intermediate and 16 high grade extrapulmonary tumors. Mean SMPL SUVmax is 8.2 ± 4.5 and SMPL/liver SUVmax is 2.4 ± 1.4. There are 37 cases (60%) with mismatched results (e.g., low FDG SMPL with intermediate or high grade extrapulmonary tumor or vice versa) and 25 matched cases (40%) that are inconclusive (e.g., low FDG with low tumor grade or high FDG with high tumor grade). Of the mismatched cases, we correctly predicted 30 cases (81%) as primary lung cancers. CONCLUSION: A mismatch between the SMPL SUVmax and the extrapulmonary tumor grade could be used to differentiate a primary lung cancer from a metastasis with reasonable accuracy. Our preliminary results support the hypothesis that FDG uptake intensity of a metastatic pulmonary lesion mirrors the tumor aggressiveness of its extrapulmonary neoplasm of origin.

Dysregulation of Lipid and Glucose Homeostasis in Hepatocyte-Specific SLC25A34 Knockout Mice.

Nonalcoholic fatty liver disease (NAFLD) is an epidemic affecting 30% of the US population. It is characterized by insulin resistance, and by defective lipid metabolism and mitochondrial dysfunction in the liver. SLC25A34 is a major repressive target of miR-122, a miR that has a central role in NAFLD and liver cancer. However, little is known about the function of SLC25A34. To investigate SLC25A34 in vitro, mitochondrial respiration and bioenergetics were examined using hepatocytes depleted of Slc25a34 or overexpressing Slc25a34. To test the function of SLC25A34 in vivo, a hepatocyte-specific knockout mouse was generated, and loss of SLC25A34 was assessed in mice maintained on a chow diet and a fast-food diet (FFD), a model for NAFLD. Hepatocytes depleted of Slc25a34 displayed increased mitochondrial biogenesis, lipid synthesis, and ADP/ATP ratio; Slc25a34 overexpression had the opposite effect. In the knockout model on chow diet, SLC25A34 loss modestly affected liver function (altered glucose metabolism was the most pronounced defect). RNA-sequencing revealed changes in metabolic processes, especially fatty acid metabolism. After 2 months on FFD, knockouts had a more severe phenotype, with increased lipid content and impaired glucose tolerance, which was attenuated after longer FFD feeding (6 months). This work thus presents a novel model for studying SLC25A34 in vivo in which SLC25A34 plays a role in mitochondrial respiration and bioenergetics during NAFLD.

Successful surgical weight loss with laparoscopic sleeve gastrectomy for morbid obesity prior to kidney transplantation.

Morbid obesity in kidney transplant (KT) candidates is associated with increased complications and graft failure. Multiple series have demonstrated rapid and significant weight loss after laparoscopic sleeve gastrectomy (LSG) in this population. Long-term and post-transplant weight evolutions are still largely unknown. A retrospective review was performed in eighty patients with end-stage kidney disease (ESKD) who underwent LSG in preparation for KT. From a median initial BMI of 43.7 kg/m2 , the median change at 1-year was -10.0 kg/m2 . Successful surgical weight loss (achieving a BMI < 35 kg/m2 or an excess body weight loss >50%) was attained in 76.3% and was associated with male gender, predialysis status, lower obesity class and lack of coronary artery disease. Thirty-one patients subsequently received a KT with a median delay of 16.7 months. Weight regain (increase in BMI of 5 kg/m2 postnadir) and recurrent obesity (weight regain + BMI > 35) remain a concern, occurring post-KT in 35.7% and 17.9%, respectively. Early LSG should be considered for morbidly obese patients with ESKD for improved weight loss outcomes. Early KT after LSG does not appear to affect short-term surgical weight loss. Candidates with a BMI of up to 45 kg/m2 can have a reasonable expectation to achieve the limit within 1 year.

PIRs mediate innate myeloid cell memory to nonself MHC molecules.

Immunological memory specific to previously encountered antigens is a cardinal feature of adaptive lymphoid cells. However, it is unknown whether innate myeloid cells retain memory of prior antigenic stimulation and respond to it more vigorously on subsequent encounters. In this work, we show that murine monocytes and macrophages acquire memory specific to major histocompatibility complex I (MHC-I) antigens, and we identify A-type paired immunoglobulin-like receptors (PIR-As) as the MHC-I receptors necessary for the memory response. We demonstrate that deleting PIR-A in the recipient or blocking PIR-A binding to donor MHC-I molecules blocks memory and attenuates kidney and heart allograft rejection. Thus, innate myeloid cells acquire alloantigen-specific memory that can be targeted to improve transplant outcomes.

DDX3X acts as a live-or-die checkpoint in stressed cells by regulating NLRP3 inflammasome.

The cellular stress response has a vital role in regulating homeostasis by modulating cell survival and death. Stress granules are cytoplasmic compartments that enable cells to survive various stressors. Defects in the assembly and disassembly of stress granules are linked to neurodegenerative diseases, aberrant antiviral responses and cancer1-5. Inflammasomes are multi-protein heteromeric complexes that sense molecular patterns that are associated with damage or intracellular pathogens, and assemble into cytosolic compartments known as ASC specks to facilitate the activation of caspase-1. Activation of inflammasomes induces the secretion of interleukin (IL)-1ß and IL-18 and drives cell fate towards pyroptosis-a form of programmed inflammatory cell death that has major roles in health and disease6-12. Although both stress granules and inflammasomes can be triggered by the sensing of cellular stress, they drive contrasting cell-fate decisions. The crosstalk between stress granules and inflammasomes and how this informs cell fate has not been well-studied. Here we show that the induction of stress granules specifically inhibits NLRP3 inflammasome activation, ASC speck formation and pyroptosis. The stress granule protein DDX3X interacts with NLRP3 to drive inflammasome activation. Assembly of stress granules leads to the sequestration of DDX3X, and thereby the inhibition of NLRP3 inflammasome activation. Stress granules and the NLRP3 inflammasome compete for DDX3X molecules to coordinate the activation of innate responses and subsequent cell-fate decisions under stress conditions. Induction of stress granules or loss of DDX3X in the myeloid compartment leads to a decrease in the production of inflammasome-dependent cytokines in vivo. Our findings suggest that macrophages use the availability of DDX3X to interpret stress signals and choose between pro-survival stress granules and pyroptotic ASC specks. Together, our data demonstrate the role of DDX3X in driving NLRP3 inflammasome and stress granule assembly, and suggest a rheostat-like mechanistic paradigm for regulating live-or-die cell-fate decisions under stress conditions.

PAX5-driven subtypes of B-progenitor acute lymphoblastic leukemia.

Recent genomic studies have identified chromosomal rearrangements defining new subtypes of B-progenitor acute lymphoblastic leukemia (B-ALL), however many cases lack a known initiating genetic alteration. Using integrated genomic analysis of 1,988 childhood and adult cases, we describe a revised taxonomy of B-ALL incorporating 23 subtypes defined by chromosomal rearrangements, sequence mutations or heterogeneous genomic alterations, many of which show marked variation in prevalence according to age. Two subtypes have frequent alterations of the B lymphoid transcription-factor gene PAX5. One, PAX5alt (7.4%), has diverse PAX5 alterations (rearrangements, intragenic amplifications or mutations); a second subtype is defined by PAX5 p.Pro80Arg and biallelic PAX5 alterations. We show that p.Pro80Arg impairs B lymphoid development and promotes the development of B-ALL with biallelic Pax5 alteration in vivo. These results demonstrate the utility of transcriptome sequencing to classify B-ALL and reinforce the central role of PAX5 as a checkpoint in B lymphoid maturation and leukemogenesis.

SCYL1 does not regulate REST expression and turnover.

A recent study identified SCYL1 as one of the components of the oncogenic STP axis, which promotes triple-negative breast cancer by regulating degradation of the REST tumor suppressor. Contrary to the findings of that study, herein we show by using 3 distinct genetic approaches that SCYL1 does not regulate REST turnover. Specifically, REST protein levels and turnover were identical in Scyl1+/+ and Scyl1-/- mouse embryonic fibroblasts. Similarly, targeted inactivation of SCYL1 in Hek293T cells by using CRIPSR-Cas9 technology did not affect REST steady-state level and turnover. Furthermore, RNA interference-mediated depletion of SCYL1 in Hek293T or MDA-MB-231 cells did not alter REST steady-state level and turnover. Together, our findings indicate that SCYL1 does not contribute to REST turnover and thus do not support a previous study suggesting a role for SCYL1 in mediating REST degradation.

Differential roles of caspase-1 and caspase-11 in infection and inflammation.

Caspase-1, also known as interleukin-1ß (IL-1ß)-converting enzyme (ICE), regulates antimicrobial host defense, tissue repair, tumorigenesis, metabolism and membrane biogenesis. On activation within an inflammasome complex, caspase-1 induces pyroptosis and converts pro-IL-1ß and pro-IL-18 into their biologically active forms. "ICE-/-" or "Casp1-/-" mice generated using 129 embryonic stem cells carry a 129-associated inactivating passenger mutation on the caspase-11 locus, essentially making them deficient in both caspase-1 and caspase-11. The overlapping and unique functions of caspase-1 and caspase-11 are difficult to unravel without additional genetic tools. Here, we generated caspase-1-deficient mouse (Casp1Null) on the C57BL/6 J background that expressed caspase-11. Casp1Null cells did not release IL-1ß and IL-18 in response to NLRC4 activators Salmonella Typhimurium and flagellin, canonical or non-canonical NLRP3 activators LPS and ATP, Escherichia coli, Citrobacter rodentium and transfection of LPS, AIM2 activators Francisella novicida, mouse cytomegalovirus and DNA, and the infectious agents Listeria monocytogenes and Aspergillus fumigatus. We further demonstrated that caspase-1 and caspase-11 differentially contributed to the host defense against A. fumigatus infection and to endotoxemia.

BOK Is a Non-canonical BCL-2 Family Effector of Apoptosis Regulated by ER-Associated Degradation.

The mitochondrial pathway of apoptosis is initiated by mitochondrial outer membrane permeabilization (MOMP). The BCL-2 family effectors BAX and BAK are thought to be absolutely required for this process. Here, we report that BCL-2 ovarian killer (BOK) is a bona fide yet unconventional effector of MOMP that can trigger apoptosis in the absence of both BAX and BAK. However, unlike the canonical effectors, BOK appears to be constitutively active and unresponsive to antagonistic effects of the antiapoptotic BCL-2 proteins. Rather, BOK is controlled at the level of protein stability by components of the endoplasmic reticulum (ER)-associated degradation pathway. BOK is ubiquitylated by the AMFR/gp78 E3 ubiquitin ligase complex and targeted for proteasomal degradation in a VCP/p97-dependent manner, which allows survival of the cell. When proteasome function, VCP, or gp78 activity is compromised, BOK is stabilized to induce MOMP and apoptosis independently of other BCL-2 proteins.

Molecular characterization of LC3-associated phagocytosis reveals distinct roles for Rubicon, NOX2 and autophagy proteins.

LC3-associated phagocytosis (LAP) is a process wherein elements of autophagy conjugate LC3 to phagosomal membranes. We characterize the molecular requirements for LAP, and identify Rubicon as being required for LAP but not autophagy. Rubicon is recruited to LAPosomes and is required for the activity of a Class III PI(3)K complex containing UVRAG but lacking ATG14 and Ambra1. This allows for the sustained localization of PtdIns(3)P, which is critical for recruitment of downstream autophagic proteins and stabilization of the NOX2 complex to produce reactive oxygen species. Both PtdIns(3)P and reactive oxygen species are required for conjugation of LC3 to LAPosomes and subsequent association with LAMP1(+) lysosomes. LAP is induced by engulfment of Aspergillus fumigatus, a fungal pathogen that commonly afflicts immunocompromised hosts, and is required for its optimal clearance in vivo. Therefore, we have identified molecules that distinguish LAP from canonical autophagy, thereby elucidating the importance of LAP in response to A. fumigatus infection.

Characterization of a family of novel cysteine- serine-rich nuclear proteins (CSRNP).

Gene array analysis has been widely used to identify genes induced during T cell activation. Our studies identified an immediate early gene that is strongly induced in response to IL-2 in mouse T cells which we named cysteine- serine-rich nuclear protein-1 (CSRNP-1). The human ortholog was previously identified as an AXIN1 induced gene (AXUD1). The protein does not contain sequence defined domains or motifs annotated in public databases, however the gene is a member of a family of three mammalian genes that share conserved regions, including cysteine- and serine-rich regions and a basic domain, they encode nuclear proteins, possess transcriptional activation domain and bind the sequence AGAGTG. Consequently we propose the nomenclature of CSRNP-1, -2 and -3 for the family. To elucidate the physiological functions of CSRNP-1, -2 and -3, we generated mice deficient for each of these genes by homologous recombination in embryonic stem cells. Although the CSRNP proteins have the hallmark of transcription factors and CSRNP-1 expression is highly induced by IL-2, deletion of the individual genes had no obvious consequences on normal mouse development, hematopoiesis or T cell functions. However, combined deficiencies cause partial neonatal lethality suggesting that the genes have redundant functions.

IFN-gamma induces apoptosis in developing mast cells.

Mast cells are critical effectors of allergic disease, and are now implicated in immune responses observed in arthritis, multiple sclerosis, and heart disease. Because of their role in inflammation, understanding how mast cells develop is of clinical importance. In this study we determined the effects of IFN-gamma on mast cell survival. Using in vitro culture of bone marrow cells in IL-3 plus stem cell factor, we found that the addition of IFN-gamma induced apoptosis, as exhibited by the presence of subdiploid DNA and caspase activation. IFN-gamma-mediated apoptosis was Stat1-dependent, and was accompanied by loss of mitochondrial membrane potential. Apoptosis was reduced in cultures of bone marrow cells derived from p53- or Bax-deficient mice, as well as H2K-Bcl-2 transgenic mice. IFN-gamma hyperresponsiveness has been shown to result in inflammatory disease and death in mice lacking the regulatory protein suppressor of cytokine signaling (SOCS)-1. Bone marrow cells from SOCS-1 knockout (KO) mice failed to give rise to viable mast cells after culture in IL-3 plus stem cell factor, with profound apoptosis occurring as the cultures matured. However, bone marrow cells lacking both SOCS-1 and IFN-gamma survived normally. This in vitro defect in mast cell development was recapitulated in vivo. SOCS-1 KO mice demonstrated a 67% decrease in peritoneal mast cell numbers relative to wild-type mice, a deficiency that was reversed in SOCS-1/IFN-gamma KO mice. These data demonstrate the potent regulatory effects of IFN-gamma on mast cell survival and show that this cytokine can elicit mast cell death in vitro and in vivo.

Stat5 tetramer formation is associated with leukemogenesis.

Activation of Stat5 is frequently found in leukemias. To study the mechanism and role of Stat5 activation, we introduced a constitutively activated Stat5a mutant, cS5F, into murine bone marrow (BM) cells. BM transplantation with cS5F-transfected cells caused development of multilineage leukemias in lethally irradiated wild-type or nonirradiated Rag2(-/-) mice. The leukemic cells showed strongly enhanced levels of cS5F tetramers but unchanged cS5F dimer levels in a DNA binding assay. Moreover, Stat5a mutants engineered to form only dimers, but not tetramers, failed to induce leukemias. In addition, Stat5 tetramers were found to accumulate in excess compared to dimers in various human leukemias. These data suggest that Stat5 tetramers are associated with leukemogenesis.

Regulation of progesterone levels during pregnancy and parturition by signal transducer and activator of transcription 5 and 20alpha-hydroxysteroid dehydrogenase.

The two highly related signal transducers and activators of transcription (Stats), Stat5a and Stat5b, are major mediators of prolactin signaling in both the mammary gland and in the ovary. Deficiencies in Stat5b, or in both Stat5a and Stat5b, result in loss of pregnancy during midgestation and are correlated with an increase in ovarian 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) and a decrease in serum progesterone, which normally declines only immediately before parturition. To determine the relative contribution of 20alpha-HSD to progesterone metabolism and Stat5 function during pregnancy and parturition, we created a 20alpha-HSD-deficient strain of mice by gene disruption. Mice deficient for 20alpha-HSD sustain high progesterone levels and display a delay in parturition of several days demonstrating that 20alpha-HSD regulates parturition downstream of the prostaglandin F2alpha receptor in an essential and nonredundant manner. Moreover, 20alpha-HSD deficiency partially corrected the abortion of pregnancies associated with Stat5b deficiency, supporting the concept that prolactin activation of Stat5b is important in suppressing 20alpha-HSD gene expression and thereby allowing the maintenance of progesterone levels that are required to sustain pregnancy.

Re-examination of the role of suppressor of cytokine signaling 1 (SOCS1) in the regulation of toll-like receptor signaling.

Suppressor of cytokine signaling 1 (SOCS1) is an obligate negative regulator of cytokine signaling and most importantly in vivo, signaling via the interferon-gamma (IFN-gamma) receptor. SOCS1, via its Src homology 2 domain, binds to phosphotyrosine residues in its targets, reducing the amplitude of signaling from cytokine receptors. SOCS1 is also implicated in blocking Toll-like receptor (TLR) signaling in macrophages activated by TLR agonists such as lipopolysaccharide (LPS), thus regulating multiple steps in the activation of innate immune responses. To rigorously test this, we isolated macrophages from Socs1-/- mice on multiple genetic backgrounds. We found no evidence that SOCS1 blocked TLR-activated pathways, endotoxin tolerance, or nitric oxide production. However, Socs1-/-;IFN-gamma-/- mice were extremely susceptible to LPS challenge, confirming previous findings. Because LPS induces IFN-beta production from macrophages, we tested whether SOCS1 regulates IFN-alpha/beta receptor signaling. We find that SOCS1 is required to inhibit IFN-alpha/beta receptor signaling in vitro. Furthermore, the absence of a single allele encoding TYK2, a JAK (Janus kinase) family member essential IFN-alpha/beta receptor signaling, rescued Socs1-/- mice from early lethality, even in the presence of IFN-gamma. We conclude that previous reports linking SOCS1 to TLR signaling are most likely due to effects on IFN-alpha/beta receptor signaling.

Glucocorticoids enhance activation of the human type II 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase gene.

Glucocorticoids indirectly alter adrenocortical steroid output through the inhibition of ACTH secretion by the anterior pituitary. However, previous studies suggest that glucocorticoids can directly affect adrenocortical steroid production. Therefore, we have investigated the ability of glucocorticoids to affect transcription of adrenocortical steroid biosynthetic enzymes. One potential target of glucocorticoid action in the adrenal is an enzyme critical for adrenocortical steroid production: 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase (3beta-HSD). Treatment of the adrenocortical cell line (H295R) with the glucocorticoid agonist dexamethasone (DEX) increased cortisol production and 3beta-HSD mRNA levels alone or in conjunction with phorbol ester. This increase in 3beta-HSD mRNA was paralleled by increases in Steroidogenic Acute Regulatory Protein (StAR) mRNA levels. The human type II 3beta-HSD promoter lacks a consensus palindromic glucocorticoid response element (GRE) but does contain a Stat5 response element (Stat5RE) suggesting that glucocorticoids could affect type II 3beta-HSD transcription via interaction with Stat5. Transfection experiments show enhancement of human type II 3beta-HSD promoter activity by coexpression of the glucocorticoid receptor (GR) and Stat5A and treatment with 100nM dexamethasone. Furthermore, removal of the Stat5RE either by truncation of the 5' flanking sequence in the promoter or introduction of point mutations to the Stat5RE abolished the ability of DEX to enhance 3beta-HSD promoter activity. These studies demonstrate the ability of glucocorticoids to directly enhance the expression of an adrenal steroidogenic enzyme gene albeit independent of a consensus palindromic glucocorticoid response element.