Systematic Analysis of Homologous Recombination Deficiency Testing in Ovarian Cancer-Development of Recommendations for Optimal Assay Performance.

Homologous recombination deficiency (HRD) assays are an important element of personalized oncology in ovarian carcinomas, but the optimal tissue requirements for these complex molecular assays remain unclear. As a result, a considerable percentage of assays are not successful, leading to suboptimal diagnoses for these patients. In this study, we have systematically analyzed tumor and tissue parameters for HRD analysis in a large cohort of real-world cancer samples. The aim of this study is to give recommendations for pathologists and gynecologic oncologists for selection of tissue samples to maximize the success rate of HRD analyses. Tumor samples from 2702 patients were sent to the Institute of Pathology of the Philipps-University Marburg between October 2020 and September 2022, of which 2654 were analyzed using the Myriad MyChoice HRD+ CDx assay. A total of 2396 of 2654 samples (90.3%) were successfully tested, of which 984 of 2396 (41.1%) were HRD positive and 1412 (58.9%) were HRD negative. Three hundred sixty-three of 2396 samples (15.2%) were BRCA1/2-mutated; 27 samples had a BRCA1/2 mutation and a genomic instability score (GIS) < 42. Twenty-two samples (0.9%) failed GIS measurement but displayed a BRCA1/2 mutation. BRCA1/2-mutated samples showed significantly (P < .0001) higher GIS values than those with a wild-type BRCA1/2 status. Tumor cell content, tumor area, and histology significantly (P < .0001) affected the probability of successfully analyzing a sample. Based on a systematic analysis of tumor cell content and tumor area, we recommend selecting patient high-grade serous ovarian cancer samples that display a tumor cell content ≥30% and a tumor area ≥0.5 cm2 (based on their hematoxylin and eosin) for HRD testing to allow for optimal chances of a successful analysis and conclusive results. Considering histologic and sample conditions, success rates of up to 98% can be achieved. Our comprehensive evaluation contributes to further standardization of recommendations on HRD testing in ovarian cancer, which will have a large impact on personalized therapeutic strategies in this highly aggressive tumor type.

Transcription Factor GLIS3: A New and Critical Regulator of Postnatal Stages of Mouse Spermatogenesis.

In this study, we identify a novel and essential role for the Krüppel-like zinc finger transcription factor GLI-similar 3 (GLIS3) in the regulation of postnatal spermatogenesis. We show that GLIS3 is expressed in gonocytes, spermatogonial stem cells (SSCs) and spermatogonial progenitors (SPCs), but not in differentiated spermatogonia and later stages of spermatogenesis or in somatic cells. Spermatogenesis is greatly impaired in GLIS3 knockout mice. Loss of GLIS3 function causes a moderate reduction in the number of gonocytes, but greatly affects the generation of SSCs/SPCs, and as a consequence the development of spermatocytes. Gene expression profiling demonstrated that the expression of genes associated with undifferentiated spermatogonia was dramatically decreased in GLIS3-deficient mice and that the cytoplasmic-to-nuclear translocation of FOXO1, which marks the gonocyte-to-SSC transition and is necessary for SSC self-renewal, is inhibited. These observations suggest that GLIS3 promotes the gonocyte-to-SSC transition and is a critical regulator of the dynamics of early postnatal spermatogenesis. Stem Cells 2016;34:2772-2783.

Estimates of Soil Bacterial Ribosome Content and Diversity Are Significantly Affected by the Nucleic Acid Extraction Method Employed.

Modern sequencing technologies allow high-resolution analyses of total and potentially active soil microbial communities based on their DNA and RNA, respectively. In the present study, quantitative PCR and 454 pyrosequencing were used to evaluate the effects of different extraction methods on the abundance and diversity of 16S rRNA genes and transcripts recovered from three different types of soils (leptosol, stagnosol, and gleysol). The quality and yield of nucleic acids varied considerably with respect to both the applied extraction method and the analyzed type of soil. The bacterial ribosome content (calculated as the ratio of 16S rRNA transcripts to 16S rRNA genes) can serve as an indicator of the potential activity of bacterial cells and differed by 2 orders of magnitude between nucleic acid extracts obtained by the various extraction methods. Depending on the extraction method, the relative abundances of dominant soil taxa, in particular Actino bacteria and Proteobacteria, varied by a factor of up to 10. Through this systematic approach, the present study allows guidelines to be deduced for the selection of the appropriate extraction protocol according to the specific soil properties, the nucleic acid of interest, and the target organisms.

Protection of primary neurons and mouse brain from Alzheimer's pathology by molecular tweezers.

Alzheimer's disease is a devastating cureless neurodegenerative disorder affecting >35 million people worldwide. The disease is caused by toxic oligomers and aggregates of amyloid ß protein and the microtubule-associated protein tau. Recently, the Lys-specific molecular tweezer CLR01 has been shown to inhibit aggregation and toxicity of multiple amyloidogenic proteins, including amyloid ß protein and tau, by disrupting key interactions involved in the assembly process. Following up on these encouraging findings, here, we asked whether CLR01 could protect primary neurons from Alzheimer's disease-associated synaptotoxicity and reduce Alzheimer's disease-like pathology in vivo. Using cell culture and brain slices, we found that CLR01 effectively inhibited synaptotoxicity induced by the 42-residue isoform of amyloid ß protein, including ∼80% inhibition of changes in dendritic spines density and long-term potentiation and complete inhibition of changes in basal synaptic activity. Using a radiolabelled version of the compound, we found that CLR01 crossed the mouse blood-brain barrier at ∼2% of blood levels. Treatment of 15-month-old triple-transgenic mice for 1 month with CLR01 resulted in a decrease in brain amyloid ß protein aggregates, hyperphosphorylated tau and microglia load as observed by immunohistochemistry. Importantly, no signs of toxicity were observed in the treated mice, and CLR01 treatment did not affect the amyloidogenic processing of amyloid ß protein precursor. Examining induction or inhibition of the cytochrome P450 metabolism system by CLR01 revealed minimal interaction. Together, these data suggest that CLR01 is safe for use at concentrations well above those showing efficacy in mice. The efficacy and toxicity results support a process-specific mechanism of action of molecular tweezers and suggest that these are promising compounds for developing disease-modifying therapy for Alzheimer's disease and related disorders.

Homologous Recombination Deficiency as an Ovarian Cancer Biomarker in a Real-World Cohort: Validation of Decentralized Genomic Profiling.

The diagnostic evaluation of homologous recombination deficiency (HRD) is central to define targeted therapy strategies for patients with ovarian carcinoma. We evaluated HRD in 514 ovarian carcinoma samples by next-generation sequencing of DNA libraries, including BRCA1/BRCA2 and 26,523 single-nucleotide polymorphisms using the standardized Myriad HRD assay, with the predefined cut point of ≥42 for a positive genomic instability score (GIS). All samples were measured in the central Myriad laboratory and in an academic molecular pathology laboratory. A positive GIS was detected in 196 (38.1%) of tumors, whereas 318 (61.9%) were GIS negative. Combining GIS and BRCA mutations, a total of 200 (38.9%) of the 514 tumors were HRD positive. A positive GIS was significantly associated with high-grade serous histology (P < 0.000001), grade 3 tumors (P = 0.001), and patient age <60 years (P = 0.0003). The concordance between both laboratories for the GIS status was 96.9% (P < 0.000001), with a sensitivity of 94.6% and a specificity of 98.4%. Concordance for HRD status was 97.1% (499 of 514 tumors). The percentage of HRD-positive tumors in our real-life cohort was similar to the proportion observed in the recently published PAOLA-1 trial, with high concordance between central and local laboratories. Our results support introduction of the standardized HRD assay in academic molecular pathology laboratories, thus broadening access to personalized oncology strategies for patients with ovarian cancer worldwide.

A systematic analysis of predicted phosphorylation sites within the human pregnane X receptor protein.

The pregnane X receptor (PXR, NR1I2) regulates the expression of genes that encode drug-metabolizing enzymes and drug transporter proteins in liver and intestine. Understanding the molecular mechanisms that modulate PXR activity is therefore critical for the development of effective therapeutic strategies. Several recent studies have implicated the activation of kinase signaling pathways in the regulation of PXR biological activity, although direct evidence and molecular mechanisms are currently lacking. We therefore sought to characterize potential phosphorylation sites within the PXR protein by use of a rational, comprehensive, and systematic site-directed mutagenesis approach to generate phosphomimetic mutations (Ser/Thr --> Asp) and phospho-deficient mutations (Ser/Thr --> Ala) at 18 predicted consensus kinase recognition sequences in the human PXR protein. Here, we identify amino acid residues Ser8, Thr57, Ser208, Ser305, Ser350, and Thr408 as being critical for biological activity of the PXR protein. Mutations at positions 57 and 408 abolish ligand-inducible PXR activity. Mutations in the extreme N terminus and in the PXR ligand-binding domain at positions Ser8, Ser305, Ser350, and Thr408 decrease the ability of PXR to form heterodimers with retinoid X receptor alpha. Mutations at positions Ser208, Ser305, Ser350, and Thr408 alter PXR-protein cofactor interactions. Finally, the subcellular localization of the PXR protein is profoundly affected by mutations at position Thr408. These data suggest that PXR activity can potentially be regulated by phosphorylation at specific amino acid residues within several predicted consensus kinase recognition sequences to differentially affect PXR biological activity.

GLIS3 binds pancreatic beta cell regulatory regions alongside other islet transcription factors.

The Krüppel-like zinc finger transcription factor Gli-similar 3 (GLIS3) plays a critical role in the regulation of pancreatic beta cells, with global Glis3 knockout mice suffering from severe hyperglycemia and dying by post-natal day 11. In addition, GLIS3 has been shown to directly regulate the early endocrine marker Ngn3, as well as Ins2 gene expression in mature beta cells. We hypothesize that GLIS3 regulates several other genes critical to beta cell function, in addition to Ins2, by directly binding to regulatory regions. We therefore generated a pancreas-specific Glis3 deletion mouse model (Glis3Δpanc) using a Pdx1-driven Cre mouse line. Roughly 20% of these mice develop hyperglycemia by 8-weeks and lose most of their insulin expression. However, this did not appear to be due to loss of the beta cells themselves, as no change in cell death was observed. Indeed, presumptive beta cells appeared to persist as PDX1+/INS-/MAFA-/GLUT2- cells. Islet RNA-seq analysis combined with GLIS3 ChIP-seq analysis revealed apparent direct regulation of a variety of diabetes related genes, including Slc2a2 and Mafa. GLIS3 binding near these genes coincided with binding for other islet-enriched transcription factors, indicating these are distinct regulatory hubs. Our data indicates that GLIS3 not only regulates insulin expression, but several additional genes critical for beta cell function.

The traditional Chinese herbal remedy tian xian activates pregnane X receptor and induces CYP3A gene expression in hepatocytes.

The pregnane X receptor (PXR, NR1I2) is a member of the nuclear receptor superfamily that is activated by a myriad of clinically used compounds and natural products. Activation of PXR in liver regulates the expression genes encoding proteins that are intimately involved in the hepatic uptake, metabolism, and elimination of toxic compounds from our bodies. PXR-mediated herb-drug interactions can have undesirable effects in patients receiving combination therapy. This can be especially important in cancer patients who self-administer over-the-counter herbal remedies together with conventional anticancer chemotherapeutics. Tian xian is a traditional Chinese herbal anticancer remedy that activates human PXR in cell-based reporter gene assays. Moreover, tian xian alters the strength of interaction between the human PXR protein and transcriptional cofactor proteins. A novel line of humanized PXR mice are described and used here to show that tian xian increases expression of Cyp3a11 in primary cultures of rodent hepatocytes. Tian xian also induces expression of CYP3A4 in primary cultures of human hepatocytes. Taken together, these data indicate that coadministration of tian xian is probably contraindicated in patients undergoing anticancer therapy with conventional chemotherapeutic agents. These data are of particular importance due to the fact that this herbal remedy is currently marketed as an adjunct therapy that reduces the side effects of conventional chemotherapy and is available without a prescription. Future studies should be conducted to determine the extent to which coadministration of this Chinese herbal remedy alters the pharmacokinetic and pharmacodynamic properties of conventional anticancer therapy.

Loss of Glis3 causes dysregulation of retrotransposon silencing and germ cell demise in fetal mouse testis.

Fetal germ cell development is regulated by an elaborate combination of cell-extrinsic and cell-intrinsic signals. Here we identify a novel role for the Krüppel-like transcription factor Gli-Similar 3 (Glis3) in male germ cell development in the mouse embryos. Glis3 is expressed in male germ cells during the brief window of time prior to initiation of piRNA-dependent retrotransposon surveillance. Disruption of Glis3 function led to a widespread reduction in retrotransposon silencing factors, aberrant retrotransposon expression and pronounced germ cell loss. Experimental induction of precocious Glis3 expression in vivo before its normal expression resulted in premature expression of several piRNA pathway members, suggesting that GLIS3 is necessary for the activation of the retrotransposon silencing programs. Our findings reveal an unexpected role for GLIS3 in the development of male germ cells and point to a central role for GLIS3 in the control of retrotransposon silencing in the fetal germline.

Bacterial endophyte communities of three agricultural important grass species differ in their response towards management regimes.

Endophytic bacteria are critical for plant growth and health. However, compositional and functional responses of bacterial endophyte communities towards agricultural practices are still poorly understood. Hence, we analyzed the influence of fertilizer application and mowing frequency on bacterial endophytes in three agriculturally important grass species. For this purpose, we examined bacterial endophytic communities in aerial plant parts of Dactylis glomerata L., Festuca rubra L., and Lolium perenne L. by pyrotag sequencing of bacterial 16S rRNA genes over two consecutive years. Although management regimes influenced endophyte communities, observed responses were grass species-specific. This might be attributed to several bacteria specifically associated with a single grass species. We further predicted functional profiles from obtained 16S rRNA data. These profiles revealed that predicted abundances of genes involved in plant growth promotion or nitrogen metabolism differed between grass species and between management regimes. Moreover, structural and functional community patterns showed no correlation to each other indicating that plant species-specific selection of endophytes is driven by functional rather than phylogenetic traits. The unique combination of 16S rRNA data and functional profiles provided a holistic picture of compositional and functional responses of bacterial endophytes in agricultural relevant grass species towards management practices.

The Effects of Cropping Regimes on Fungal and Bacterial Communities of Wheat and Faba Bean in a Greenhouse Pot Experiment Differ between Plant Species and Compartment.

Many bacteria and fungi in the plant rhizosphere and endosphere are beneficial to plant nutrient acquisition, health, and growth. Although playing essential roles in ecosystem functioning, our knowledge about the effects of multiple cropping regimes on the plant microbiome and their interactions is still limited. Here, we designed a pot experiment simulating different cropping regimes. For this purpose, wheat and faba bean plants were grown under controlled greenhouse conditions in monocultures and in two intercropping regimes: row and mixed intercropping. Bacterial and fungal communities in bulk and rhizosphere soils as well as in the roots and aerial plant parts were analyzed using large-scale metabarcoding. We detected differences in microbial richness and diversity between the cropping regimes. Generally, observed effects were attributed to differences between mixed and row intercropping or mixed intercropping and monoculture. Bacterial and fungal diversity were significantly higher in bulk soil samples of wheat and faba bean grown in mixed compared to row intercropping. Moreover, microbial communities varied between crop species and plant compartments resulting in different responses of these communities toward cropping regimes. Leaf endophytes were not affected by cropping regime but bacterial and fungal community structures in bulk and rhizosphere soil as well as fungal community structures in roots. We further recorded highly complex changes in microbial interactions. The number of negative inter-domain correlations between fungi and bacteria decreased in bulk and rhizosphere soil in intercropping regimes compared to monocultures due to beneficial effects. In addition, we observed plant species-dependent differences indicating that intra- and interspecific competition between plants had different effects on the plant species and thus on their associated microbial communities. To our knowledge, this is the first study investigating microbial communities in different plant compartments with respect to multiple cropping regimes using large-scale metabarcoding. Although a simple design simulating different cropping regimes was used, obtained results contribute to the understanding how cropping regimes affect bacterial and fungal communities and their interactions in different plant compartments. Nonetheless, we need field experiments to properly quantify observed effects in natural ecosystems.

GLIS3 is indispensable for TSH/TSHR-dependent thyroid hormone biosynthesis and follicular cell proliferation.

Deficiency in Krüppel-like zinc finger transcription factor GLI-similar 3 (GLIS3) in humans is associated with the development of congenital hypothyroidism. However, the functions of GLIS3 in the thyroid gland and the mechanism by which GLIS3 dysfunction causes hypothyroidism are unknown. In the current study, we demonstrate that GLIS3 acts downstream of thyroid-stimulating hormone (TSH) and TSH receptor (TSHR) and is indispensable for TSH/TSHR-mediated proliferation of thyroid follicular cells and biosynthesis of thyroid hormone. Using ChIP-Seq and promoter analysis, we demonstrate that GLIS3 is critical for the transcriptional activation of several genes required for thyroid hormone biosynthesis, including the iodide transporters Nis and Pds, both of which showed enhanced GLIS3 binding at their promoters. The repression of cell proliferation of GLIS3-deficient thyroid follicular cells was due to the inhibition of TSH-mediated activation of the mTOR complex 1/ribosomal protein S6 (mTORC1/RPS6) pathway as well as the reduced expression of several cell division-related genes regulated directly by GLIS3. Consequently, GLIS3 deficiency in a murine model prevented the development of goiter as well as the induction of inflammatory and fibrotic genes during chronic elevation of circulating TSH. Our study identifies GLIS3 as a key regulator of TSH/TSHR-mediated thyroid hormone biosynthesis and proliferation of thyroid follicular cells and uncovers a mechanism by which GLIS3 deficiency causes neonatal hypothyroidism and prevents goiter development.

Driving forces of soil bacterial community structure, diversity, and function in temperate grasslands and forests.

Soil bacteria provide a large range of ecosystem services such as nutrient cycling. Despite their important role in soil systems, compositional and functional responses of bacterial communities to different land use and management regimes are not fully understood. Here, we assessed soil bacterial communities in 150 forest and 150 grassland soils derived from three German regions by pyrotag sequencing of 16S rRNA genes. Land use type (forest and grassland) and soil edaphic properties strongly affected bacterial community structure and function, whereas management regime had a minor effect. In addition, a separation of soil bacterial communities by sampling region was encountered. Soil pH was the best predictor for bacterial community structure, diversity and function. The application of multinomial log-linear models revealed distinct responses of abundant bacterial groups towards pH. Predicted functional profiles revealed that differences in land use not only select for distinct bacterial populations but also for specific functional traits. The combination of 16S rRNA data and corresponding functional profiles provided comprehensive insights into compositional and functional adaptations to changing environmental conditions associated with differences in land use and management.

Fine Spatial Scale Variation of Soil Microbial Communities under European Beech and Norway Spruce.

The complex interactions between trees and soil microbes in forests as well as their inherent seasonal and spatial variations are poorly understood. In this study, we analyzed the effects of major European tree species (Fagus sylvatica L. and Picea abies (L.) Karst) on soil bacterial and fungal communities. Mineral soil samples were collected from different depths (0-10, 10-20 cm) and at different horizontal distances from beech or spruce trunks (0.5, 1.5, 2.5, 3.5 m) in early summer and autumn. We assessed the composition of soil bacterial and fungal communities based on 16S rRNA gene and ITS DNA sequences. Community composition of bacteria and fungi was most strongly affected by soil pH and tree species. Different ectomycorrhizal fungi (e.g., Tylospora) known to establish mutualistic associations with plant roots showed a tree species preference. Moreover, bacterial and fungal community composition showed spatial and seasonal shifts in soil surrounding beech and spruce. The relative abundance of saprotrophic fungi was higher at a depth of 0-10 vs. 10-20 cm depth. This was presumably a result of changes in nutrient availability, as litter input and organic carbon content decreased with soil depth. Overall bacterial community composition showed strong variations under spruce with increasing distance from the tree trunks, which might be attributed in part to higher fine root biomass near spruce trunks. Furthermore, overall bacterial community composition was strongly affected by season under deciduous trees.

TRANSCRIPTION FACTOR GLI-SIMILAR 3 (GLIS3): IMPLICATIONS FOR THE DEVELOPMENT OF CONGENITAL HYPOTHYROIDISM.

Congenital hypothyroidism (CH) is the most frequent endocrine disorder in neonates. While several genetic mutations have been identified that result in developmental defects of the thyroid gland or thyroid hormone synthesis, genetic factors have yet to be identified in many CH patients along with the mechanisms underlying their pathophysiology. Mutations in the gene encoding the Krüppel-like transcription factor, GLI-similar 3 (GLIS3) have been associated with the development of a syndrome characterized by congenital hypothyroidism and neonatal diabetes and similar phenotypes were observed in mouse knockout models of Glis3. Patients with GLIS3-mediated CH exhibit diminished serum levels of thyroxine (T4) and triiodothyronine (T3) and elevated thyroid stimulating hormone (TSH) and thyroglobulin (TG). However, the inconsistent presentation of clinical features associated with this CH has made it difficult to ascertain a causative mechanism. Future elucidation of the biological functions of GLIS3 in the thyroid will be crucial to the discovery of new therapeutic opportunities for the treatment of CH.

Gli-similar proteins: their mechanisms of action, physiological functions, and roles in disease.

Gli-similar (Glis) 1-3 proteins constitute a subfamily of Krüppel-like zinc-finger proteins that are closely related to members of the Gli family. Glis proteins have been implicated in several pathologies, including cystic kidney disease, diabetes, hypothyroidism, fibrosis, osteoporosis, psoriasis, and cancer. In humans, a mutation in the Glis2 gene has been linked to the development of nephronophthisis (NPHP), a recessive cystic kidney disease, while mutations in Glis3 lead to an extended multisystem phenotype that includes the development of neonatal diabetes, polycystic kidneys, congenital hypothyroidism, and facial dysmorphism. Glis3 has also been identified as a risk locus for type-1 and type-2 diabetes and additional studies have revealed a role for Glis3 in pancreatic endocrine development, ß-cell maintenance, and insulin regulation. Similar to Gli1-3, Glis2 and 3 have been reported to localize to the primary cilium. These studies appear to suggest that Glis proteins are part of a primary cilium-associated signaling pathway(s). It has been hypothesized that Glis proteins are activated through posttranslational modifications and subsequently translocate to the nucleus where they regulate transcription by interacting with Glis-binding sites in the promoter regions of target genes. This chapter summarizes the current state of knowledge regarding mechanisms of action of the Glis family of proteins, their physiological functions, as well as their roles in disease.

Gli-similar (Glis) Krüppel-like zinc finger proteins: insights into their physiological functions and critical roles in neonatal diabetes and cystic renal disease.

GLI-similar (Glis) 1-3 proteins constitute a subfamily of the Krüppel-like zinc finger transcription factors that are closely related to the Gli family. Glis1-3 play critical roles in the regulation of a number of physiological processes and have been implicated in several pathologies. Mutations in GLIS2 have been linked to nephronophthisis, an autosomal recessive cystic kidney disease. Loss of Glis2 function leads to renal atrophy and fibrosis that involves epithelial-mesenchymal transition (EMT) of renal tubule epithelial cells. Mutations in human GLIS3 have been implicated in a syndrome characterized by neonatal diabetes and congenital hypothyroidism (NDH) and in some patients accompanied by polycystic kidney disease, glaucoma, and liver fibrosis. In addition, the GLIS3 gene has been identified as a susceptibility locus for the risk of type 1 and 2 diabetes. Glis3 plays a key role in pancreatic development, particularly in the generation of ß-cells and in the regulation of insulin gene expression. Glis2 and Glis3 proteins have been demonstrated to localize to the primary cilium, a signaling organelle that has been implicated in several pathologies, including cystic renal diseases. This association suggests that Glis2/3 are part of primary cilium-associated signaling pathways that control the activity of Glis proteins. Upon activation in the primary cilium, Glis proteins may translocate to the nucleus where they subsequently regulate gene transcription by interacting with Glis-binding sites in the promoter regulatory region of target genes. In this review, we discuss the current knowledge of the Glis signaling pathways, their physiological functions, and their involvement in several human pathologies.

Cyclic AMP-dependent protein kinase signaling modulates pregnane x receptor activity in a species-specific manner.

Pregnane x receptor is a ligand-activated transcription factor that regulates drug-inducible expression of several key cytochrome P450 enzymes and drug transporter proteins in liver and intestine in a species-specific manner. Activation of this receptor modulates several key biochemical pathways, including gluconeogenesis, beta-oxidation of fatty acids, fatty acid uptake, cholesterol homeostasis, and lipogenesis. It is of current interest to determine whether the interaction between pregnane x receptor and these key biochemical pathways is evolutionarily conserved. We show here that activation of the cyclic AMP-dependent protein kinase signaling pathway synergizes with pregnane x receptor-mediated gene activation in mouse hepatocytes. Conversely, cyclic AMP-dependent protein kinase signaling has a repressive effect upon pregnane x receptor-mediated gene activation in rat and human hepatocytes. We show that the human pregnane x receptor protein can serve as an effective substrate for catalytically active cyclic AMP-dependent protein kinase in vitro. Metabolic labeling of the protein in vivo indicates that human pregnane x receptor exists as a phosphoprotein and that activation of cyclic AMP-dependent protein kinase signaling modulates the phosphorylation status of pregnane x receptor. Activation of cyclic AMP-dependent protein kinase signaling also modulates the interactions of pregnane x receptor with protein cofactors. Our results define the species-specific impact of cyclic AMP-dependent protein kinase signaling on pregnane x receptor and provide a molecular explanation of cyclic AMP-dependent protein kinase-mediated repression of human pregnane x receptor activity. Taken together, our results identify a novel mode of regulation of pregnane x receptor activity and highlight prominent functional differences in the process across species.