Helicase-like transcription factor (HLTF)-deleted CDX/TME model of colorectal cancer increased transcription of oxidative phosphorylation genes and diverted glycolysis to boost S-glutathionylation in lymphatic intravascular metastatic niches.

Helicase-like transcription factor (HLTF) also known as SMARCA3, protects genome integrity. A tumor suppressor, HLTF is expressed in tumor cells but not in the tumor microenvironment (TME) in early-stage colorectal cancer (CRC). With disease progression, there is high concordance between epigenetic silencing of HLTF in CRC cells and negligible HLTF expression in the TME. We developed a cell line-derived xenograft (CDX) model and show for the first time that HLTF-deletion in cancer cells and the TME results in metabolic reprogramming that mitigates oxidative stress in lymphatic intravascular metastatic niches. The two metabolic pathways that derive energy from glucose-glycolysis and oxidative phosphorylation (OXPHOS)-are variously utilized by cancer cells depending upon the TME. HIF-1α, a master regulator of glycolysis, was eliminated from a role in reprogramming metabolism to satisfy CDX energetic requirements by RNAseq and spatial transcriptomics. Variability in the gut microbiome, with a putative role in altered metabolism, was also eliminated. HLTF-deleted cancer cells recovered from DNA damage at a transcriptomic level induction of DNA repair and OXPHOS genes linked to an amoeboid-associated phenotype at the tumor border (confocal microscopy). HLTF-deleted cancer and endothelial cells of lymphatic (PDPN) intravascular niches in the TME shared a site-specific protein S-glutathionylation signature (2D DIGE, MALDI-TOF/TOF mass spectrometry) for three glycolytic enzymes (PGK1 Cys379/380, PGAM1 Cys55, ENOA1 Cys119) that diverted glycolysis in support of continued glutathione biosynthesis. The collective absence of HLTF/Hltf from tumor and TME achieved redox homeostasis throughout the CDX and promoted metastasis.

Helicase-like transcription factor (Hltf)-deletion activates Hmgb1-Rage axis and granzyme A-mediated killing of pancreatic ß cells resulting in neonatal lethality.

Epigenetic mechanisms are integral to pancreatic ß cell function. Promoter hypermethylation of the helicase like-transcription factor (HLTF) gene-a component of the cellular DNA damage response that contributes to genome stability-has been implicated in age-associated changes in ß cells. To study HLTF, we generated global and ß cell-specific (ß) Hltf knockout (KO) immune competent (IC) and immune deficient (ID) Rag2-/IL2- mice. IC global and ß Hltf KO mice were neonatal lethal whereas ID global and ß Hltf KO newborn mice had normal survival. This focused our investigation on the effects of Rag2 interruption with common gamma chain interruption on ß cell function/survival. Three-way transcriptomic (RNAseq) analyses of whole pancreata from IC and ID newborn ß Hltf KO and wild type (Hltf +/+) controls combined with spatially resolved transcriptomic analysis of formalin fixed paraffin embedded tissue, immunohistochemistry and laser scanning confocal microscopy showed DNA damage caused by ß Hltf KO in IC mice upregulated the Hmgb1-Rage axis and a gene signature for innate immune cells. Perforin-delivered granzyme A (GzmA) activation of DNase, Nme1, showed damaged nuclear single-stranded DNA (γH2AX immunostaining). This caspase-independent method of cell death was supported by transcriptional downregulation of Serpinc1 gene that encodes a serine protease inhibitor of GzmA. Increased transcriptional availability of complement receptors C3ar1 and C5ar1 likely invited crosstalk with Hmgb1 to amplify inflammation. This study explores the complex dialog between ß cells and immune cells during development. It has implications for the initiation of type I diabetes in utero when altered gene expression that compromises genome stability invokes a localized inflammatory response.

Sertoli Cells Express Accommodation, Survival, and Immunoregulatory Factors When Exposed to Normal Human Serum.

Transplantation is a clinical procedure that treats a variety of diseases yet is unattainable for many patients due to a nationwide organ shortage and the harsh side effects of chronic immune suppression. Xenografted pig organs are an attractive alternative to traditional allografts and would provide an endless supply of transplantable tissue, but transplants risk rejection by the recipient's immune system. An essential component of the rejection immune response is the complement system. Sertoli cells, an immunoregulatory testicular cell, survive complement as xenografts long term without any immune suppressants. We hypothesized that exposure to the xenogeneic complement influences Sertoli cell gene expression of other accommodation factors that contribute to their survival; thus, the purpose of this study was to describe these potential changes in gene expression. RNA sequencing of baseline neonatal pig Sertoli cells (NPSC) as compared to NPSC after exposure to normal human serum (NHS, containing complement) revealed 62 significantly differentially expressed genes (DEG) that affect over 30 pathways involved in immune regulation, cell survival, and transplant accommodation. Twelve genes of interest were selected for further study, and Sertoli cell protein expression of CCL2 and the accommodation factor A20 were confirmed for the first time. Functional pathway analyses were conducted in NPSC and three biological clusters were revealed as being considerably affected by NHS exposure: innate immune signaling, cytokine signaling, and T cell regulation. Better understanding of the interaction of Sertoli cells with complement in a xenograft environment may reveal the mechanisms behind immune-privileged systems to increase graft viability.

The Sertoli Cell Complement Signature: A Suspected Mechanism in Xenograft Survival.

The complement system is an important component of transplant rejection. Sertoli cells, an immune regulatory testicular cell, survive long-term when transplanted across immunological barriers; thus, understanding the mechanisms behind this unique survival would be of great benefit to the transplantation field. This study focused on Sertoli cell inhibition of complement as relevant in xenotransplantation. Neonatal pig Sertoli cells (NPSCs) survived activated human complement in vitro while neonatal pig islet (NPI) aggregates and pig aortic endothelial cell (PAEC) survival were diminished to about 65% and 12%, respectively. PAECs cultured in NPSC-conditioned media and human complement demonstrated a 200% increase in survival suggesting that NPSCs secrete complement-inhibiting substances that confer protection. Bioinformatic and molecular analyses identified 21 complement inhibitors expressed by NPSCs with several significantly increased in NPSCs compared to NPIs or PAECs. Lastly, RNA sequencing revealed that NPSCs express 25 other complement factors including cascade components and receptors. Overall, this study identified the most comprehensive Sertoli cell complement signature to date and indicates that the expression of a variety of complement inhibitors ensures a proper regulation of complement through redundant inhibition points. Understanding the regulation of the complement system should be further investigated for extending xenograft viability.

Helicase-like transcription factor-deletion from the tumor microenvironment in a cell line-derived xenograft model of colorectal cancer reprogrammed the human transcriptome-S-nitroso-proteome to promote inflammation and redirect metastasis.

Methylation of the HLTF gene in colorectal cancer (CRC) cells occurs more frequently in men than women. Progressive epigenetic silencing of HLTF in tumor cells is accompanied by negligible expression in the tumor microenvironment (TME). Cell line-derived xenografts (CDX) were established in control (Hltf+/+) and Hltf-deleted male Rag2-/-IL2rg-/- mice by direct orthotopic cell microinjection (OCMI) of HLTF+/+HCT116 Red-FLuc cells into the submucosa of the cecum. Combinatorial induction of IL6 and S100A8/A9 in the Hltf-deleted TME with ICAM-1 and IL8 in the primary tumor activated a positive feedback loop. The proinflammatory niche produced a major shift in CDX metastasis to peritoneal dissemination compared to controls. Inducible nitric oxide (iNOS) gene expression and transactivation of the iNOS-S100A8/A9 signaling complex in Hltf-deleted TME reprogrammed the human S-nitroso-proteome. POTEE, TRIM52 and UN45B were S-nitrosylated on the conserved I/L-X-C-X2-D/E motif indicative of iNOS-S100A8/A9-mediated S-nitrosylation. 2D-DIGE and protein identification by MALDI-TOF/TOF mass spectrometry authenticated S-nitrosylation of 53 individual cysteines in half-site motifs (I/L-X-C or C-X-X-D/E) in CDX tumors. POTEE in CDX tumors is both a general S-nitrosylation target and an iNOS-S100A8/A9 site-specific (Cys638) target in the Hltf-deleted TME. REL is an example of convergence of transcriptomic-S-nitroso-proteomic signaling. The gene is transcriptionally activated in CDX tumors with an Hltf-deleted TME, and REL-SNO (Cys143) was found in primary CDX tumors and all metastatic sites. Primary CDX tumors from Hltf-deleted TME shared 60% of their S-nitroso-proteome with all metastatic sites. Forty percent of SNO-proteins from primary CDX tumors were variably expressed at metastatic sites. Global S-nitrosylation of proteins in pathways related to cytoskeleton and motility was strongly implicated in the metastatic dissemination of CDX tumors. Hltf-deletion from the TME played a major role in the pathogenesis of inflammation and linked protein S-nitrosylation in primary CDX tumors with spatiotemporal continuity in metastatic progression when the tumor cells expressed HLTF.

Helicase-like transcription factor (Hltf) gene-deletion promotes oxidative phosphorylation (OXPHOS) in colorectal tumors of AOM/DSS-treated mice.

The helicase-like transcription factor (HLTF) gene-a tumor suppressor in human colorectal cancer (CRC)-is regulated by alternative splicing and promoter hypermethylation. In this study, we used the AOM/DSS-induced mouse model to show Hltf-deletion caused poor survival concomitant with increased tumor multiplicity, and dramatically shifted the topographic distribution of lesions into the rectum. Differential isoform expression analysis revealed both the truncated isoform that lacks a DNA-repair domain and the full length isoform capable of DNA damage repair are present during adenocarcinoma formation in controls. iPathwayGuide identified 51 dynamically regulated genes of 10,967 total genes with measured expression. Oxidative Phosphorylation (Kegg: 00190), the top biological pathway perturbed by Hltf-deletion, resulted from increased transcription of Atp5e, Cox7c, Uqcr11, Ndufa4 and Ndufb6 genes, concomitant with increased endogenous levels of ATP (p = 0.0062). Upregulation of gene expression, as validated with qRT-PCR, accompanied a stable mtDNA/nDNA ratio. This is the first study to show Hltf-deletion in an inflammation-associated CRC model elevates mitochondrial bioenergetics.

Alternative splicing of helicase-like transcription factor (Hltf): Intron retention-dependent activation of immune tolerance at the feto-maternal interface.

Hltf is regulated by intron retention, and global Hltf-deletion causes perinatal lethality from hypoglycemia. In heart, full-length Hltf is a transcriptional regulator of Hif-1α that controls transport systems. Thus, we tested the hypothesis that Hltf deletion from placenta caused or exacerbated neonatal hypoglycemia via Hif-1α regulation of nutrient transporters. RNA-seq data analyses identified significant changes in transcript expression and alternative splicing (AS) in E18.5 placentome. iPathwayGuide was used for gene ontology (GO) analysis of biological processes, molecular functions and cellular components. Elim pruning algorithm identified hierarchical relationships. The methylome was interrogated by Methyl-MiniSeq Epiquest analysis. GO analysis identified gene enrichment within biological processes. Protein expression was visualized with immunohistochemistry. Although two Hltf mRNA isoforms are quantifiable in most murine tissues, only the truncated Hltf isoform is expressed in placenta. The responsible intron retention event occurs in the absence of DNA methylation. iPathwayGuide analysis identified 157 target genes of 11,538 total genes with measured expression. These were obtained using a threshold of 0.05 for statistical significance (p-value) and a long fold change of expression with absolute value of at least 0.6. Hltf deletion altered transcription of trophoblast lineage-specific genes, and increased transcription of the Cxcr7 (p = 0.004) gene whose protein product is a co-receptor for human and simian immunodeficiency viruses. Concomitant increased Cxcr7 protein was identified with immunolabeling. Hltf deletion had no effect on transcription or site-specific methylation patterns of Hif-1α, the major glucose transporters, or System A amino acid transporters. There was no measureable evidence of uteroplacental dysfunction or fetal compromise. iPathGuide analysis revealed Hltf suppresses cytolysis (10/21 genes; p-value 1.900e-12; p-value correction: Elim pruning; GO:019835) including the perforin-granzyme pathway in uterine natural killer cells. Our findings 1) prove the truncated Hltf protein isoform is a transcription factor, 2) establish a functional link between AS of Hltf and immunosuppression at the feto-maternal interface, 3) correlate intron retention with the absence of DNA methylation, and 4) underscore the importance of differential splicing analysis to identify Hltf's functional diversity.

Helicase-like transcription factor (Hltf) regulates G2/M transition, Wt1/Gata4/Hif-1a cardiac transcription networks, and collagen biogenesis.

HLTF/Hltf regulates transcription, remodels chromatin, and coordinates DNA damage repair. Hltf is expressed in mouse brain and heart during embryonic and postnatal development. Silencing Hltf is semilethal. Seventy-four percent of congenic C57BL/6J Hltf knockout mice died, 75% within 12-24 hours of birth. Previous studies in neonatal (6-8 hour postpartum) brain revealed silencing Hltf disrupted cell cycle progression, and attenuated DNA damage repair. An RNA-Seq snapshot of neonatal heart transcriptome showed 1,536 of 20,000 total transcripts were altered (p < 0.05) - 10 up- and 1,526 downregulated. Pathway enrichment analysis with MetaCore™ showed Hltf's regulation of the G2/M transition (p=9.726E(-15)) of the cell cycle in heart is nearly identical to its role in brain. In addition, Brca1 and 12 members of the Brca1 associated genome surveillance complex are also downregulated. Activation of caspase 3 coincides with transcriptional repression of Bcl-2. Hltf loss caused downregulation of Wt1/Gata4/Hif-1a signaling cascades as well as Myh7b/miR499 transcription. Hltf-specific binding to promoters and/or regulatory regions of these genes was authenticated by ChIP-PCR. Hif-1a targets for prolyl (P4ha1, P4ha2) and lysyl (Plod2) collagen hydroxylation, PPIase enzymes (Ppid, Ppif, Ppil3) for collagen trimerization, and lysyl oxidase (Loxl2) for collagen-elastin crosslinking were downregulated. However, transcription of genes for collagens, fibronectin, Mmps and their inhibitors (Timps) was unaffected. The collective downregulation of genes whose protein products control collagen biogenesis caused disorganization of the interstitial and perivascular myocardial collagen fibrillar network as viewed with picrosirius red-staining, and authenticated with spectral imaging. Wavy collagen bundles in control hearts contrasted with collagen fibers that were thin, short and disorganized in Hltf null hearts. Collagen bundles in Hltf null hearts were tangled and fragmented. Thus, silencing Hltf during heart organogenesis compromised DNA double-strand break repair, and caused aberrant collagen biogenesis altering the structural network that transmits cardiomyocyte force into muscle contraction.

Role of helicase-like transcription factor (hltf) in the G2/m transition and apoptosis in brain.

HLTF participates in transcription, chromatin remodeling, DNA damage repair, and tumor suppression. Aside from being expressed in mouse brain during embryonic and postnatal development, little is known about Hltf's functional importance. Splice variant quantification of wild-type neonatal (6-8 hour postpartum) brain gave a ratio of 5:1 for Hltf isoform 1 (exons 1-25) to isoform 2 (exons 1-21 with exon 21 extended via a partial intron retention event). Western analysis showed a close correlation between mRNA and protein expression. Complete loss of Hltf caused encephalomalacia with increased apoptosis, and reduced viability. Sixty-four percent of Hltf null mice died, 48% within 12-24 hours of birth. An RNA-Seq snapshot of the neonatal brain transcriptome showed 341 of 20,000 transcripts were altered (p < 0.05) - 95 up regulated and 246 down regulated. MetaCore™ enrichment pathway analysis revealed Hltf regulates cell cycle, cell adhesion, and TGF-beta receptor signaling. Hltf's most important role is in the G2/M transition of the cell cycle (p  =  4.672e-7) with an emphasis on transcript availability of major components in chromosome cohesion and condensation. Hltf null brains have reduced transcript levels for Rad21/Scc1, histone H3.3, Cap-E/Smc2, Cap-G/G2, and Aurora B kinase. The loss of Hltf in its yeast Rad5-like role in DNA damage repair is accompanied by down regulation of Cflar, a critical inhibitor of TNFRSF6-mediated apoptosis, and increased (p<0.0001) active caspase-3, an indicator of intrinsic triggering of apoptosis in null brains. Hltf also regulates Smad7/Bambi/Tgf-beta/Bmp5/Wnt10b signaling in brain. ChIP confirmed Hltf binding to consensus sequences in predicted (promoter Scgb3a1 gene) and previously unidentified (P-element on chromosome 7) targets. This study is the first to provide a comprehensive view of Hltf targets in brain. Moreover, it reveals how silencing Hltf disrupts cell cycle progression, and attenuates DNA damage repair.

Prolactin induces Jak2 phosphorylation of RUSHY195.

Jak2/RUSH-mediated prolactin signaling culminates in RUSH-1α-DNA-binding. Heretofore, Jak2-specific phosphorylation residues in RUSH were unknown. Genpathway's discovery approaches correlated RUSH-DNA binding (-126/-121) in uteroglobin's proximal promoter with recruitment of the transcriptional machinery. NetPhos 2.0 server found a single tyrosine phosphorylation site in RUSH's minimal DNA-binding domain. Y195 had identical context and prediction scores (0.52) for rabbit and human (HLTF) orthologs. The mouse ortholog (Hltf) had a higher prediction score (0.897). Affinity purified RUSHY195ph antibodies recognized native tyrosine phosphorylated RUSH protein immunoprecipitated from nuclear extracts. When R5020-treated HRE-H9 cells±the Jak2 inhibitor, Tyrene CR4, were stimulated with prolactin, confocal immunofluorescence images provided conclusive evidence that Jak2 mediated the availability of phosphorylated RUSHY195 in nucleus and cytoplasm. Catalytically active Jak2 is ipso facto a RUSH site-specific tyrosine kinase. Immunoprecipitation/Western blotting revealed both phosphorylation at Y195 and the physical interaction between p-Jak2/RUSH/HLTF/Hltf are evolutionarily conserved across three mammalian (rabbit, human, mouse) orthologs.

Prolactin-induced Jak2 phosphorylation of RUSH: a key element in Jak/RUSH signaling.

Jak2/Stat-mediated prolactin signaling culminates in Stat5a-DNA-binding. However, not all Jak2-dependent genes have Stat5 sites. Western analysis with inhibitors showed Jak2 is a proximal intermediate in prolactin-induced RUSH phosphorylation. Transfection assays with HRE-H9 cells showed the RUSH-binding site mediated the ability of prolactin to augment progesterone-dependent transcription of the RUSH gene. Jak2 inhibitors or targeted RUSH-site mutation blocked the prolactin effect. RUSH co-immunoprecipitated with phospho-Jak2 from nuclear extracts. Jak2 inhibitors abolished the nuclear pool of phospho-RUSH not the nuclear content of RUSH in HRE-H9 cells. Nucleolar-affiliated partners, e.g. nucleolin, were identified by microLC/MS/MS analysis of nuclear proteins that co-immunoprecipitated with RUSH/GST-RING. RUSH did not exclusively co-localize with fibrillarin to the nucleolus. MG-132 (proteasomal inhibitor) failed to block Tyrene CR4-mediated decrease in phospho-RUSH, and did not promote RUSH accumulation in the nucleolus. These studies authenticate prolactin-dependent Jak2 phosphorylation of RUSH, and provide functional implications on the RUSH network of nuclear interactions.

Conservation of inter-protein binding sites in RUSH and RFBP, an ATP11B isoform.

Isoforms of RUSH interact with a RING-finger binding protein (RFBP), which is a splice variant of the Type IV P-type ATPase, ATP11B. Splice arrays and RT-PCR showed that although most splice variants in RUSH and ATP11B are conserved in human and rabbit, the RFBP isoform is specific to rabbit. Interactions between the discontinuous PVITHC-HAKCPL sequence in the RING-domain of RUSH and the KVIRLIKIS sequence in the catalytic loop of RFBP were first identified with pull-down assays. Fine mapping involved probing CLIPS-constrained RING peptides with GST-tagged KVIRLIKIS. When the companion site in RFBP was fine mapped by replacement analysis with MBP-tagged RING, a four-fold increase in binding was noted for the KVIRLDKIS mutant. Direct comparison of splicing events in the RUSH and ATP11B genes between human and rabbit shows high structural stability in these protein interactions sites, which are 100% conserved in all mammalian orthologs.

Progesterone regulation of RUSH/SMARCA3/HLTF includes DNA looping.

RUSH/SMARCA3 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A, member 3) is capable of sequence-selective DNA binding and ATP-dependent DNA unwinding. In rabbit uterine epithelial cells, RUSH-1alpha (113 kDa) is the progesterone-dependent splice variant and RUSH-1beta (95 kDa) is the oestrogen-dependent splice variant. Rabbit RUSH/SMARCA3 mRNA is primarily regulated at the proximal promoter (-162/+90) via a PRE (progesterone-response element) half-site/overlapping Y-box domain (-38/-26) and two Sp (specificity protein) 3 sites centred at -128 and -58. We investigated hormone regulation by exploring binding of transcription factors to a putative RUSH/SMARCA3 site (-616/-611) and the distal Sp3 (-131/-126) site. In response to progesterone, RUSH-1alpha binds the RUSH site and the Sp3 site becomes a functional binding site for Egr-1 (early growth-response gene product 1)/Sp (specificity protein)1/3/MAZ (Myc-associated zinc-finger protein)/MZF1 (myeloid zinc finger 1)/c-Rel. TransSignal TF-TF Interaction Arrays, supershift assays and ChIP (chromatin immunoprecipitation) analyses confirmed strong physical interactions between RUSH and Egr-1/c-Rel. Higher-order long-range interactions between RUSH and the Egr-1/c-Rel derivative of the anisotropic flexibility of the intervening DNA sequence were shown with 3C (chromosome conformation capture) assays. Transient transfection assays with mutant constructs showed the co-operative interaction between RUSH and Egr-1 mediates repression by c-Rel. Thus DNA-bound RUSH/SMARCA3 communicates with its own proximal promoter by looping the intervening DNA. Moreover, progesterone-dependent DNA looping is an adjunct to progesterone induction of the RUSH/SMARCA3 gene because the availability of RUSH isoforms and relevant binding partners is progesterone-regulated.

Progesterone-dependent deoxyribonucleic acid looping between RUSH/SMARCA3 and Egr-1 mediates repression by c-Rel.

Steroids regulate alternative splicing of RUSH/SMARCA3. The full-length, progesterone-dependent alpha-isoform and the 3'-truncated, estrogen-dependent beta-isoform have identical DNA-binding domains, nuclear localization signals, and RING fingers. Transcription of RUSH/SMARCA3 is mediated by a bipartite progesterone receptor half-site/overlapping Y-box combination (-38/-26), where progesterone activation is attenuated by nuclear factor Y binding. Regulation also involves two GC-rich sequences in the proximal promoter (-162/+90) and a RUSH/SMARCA3 site (-616/-611) in the 5'-untranslated region. Isoform-specific binding to the RUSH/SMARCA3 site is dictated by the hormonal milieu, as is the availability of factors that bind to the distal GC-rich site (-131/-126), a composite binding site for Egr-1/specific protein-1/3/Myc-associated zinc finger protein/myeloid zinc finger-1/c-Rel, and the proximal GC-rich site (-62/-53), which binds only Sp1/3. TransSignal TF-TF interaction arrays, supershift assays, and chromatin immunoprecipitation analyses confirmed strong physical interactions between RUSH/Egr-1 and RUSH/c-Rel that were visualized with fluorescent microscopy. Higher-order, long-range interactions between RUSH and Egr-1/c-Rel derivative of the anisotropic flexibility of the intervening DNA sequence were shown by Chromosome Conformation Capture assay. Glutathione S-transferase pull-downs confirmed that the RING finger is the protein-binding domain, suggesting that the RUSH isoforms have equivalent potential for protein interactions. Transient transfection assays showed that the cooperative interaction between RUSH and Egr-1 mediates repression by c-Rel. Thus, progesterone-induced transcription is fine-tuned by isoform-specific autoregulation, in which newly synthesized RUSH-1alpha binds DNA and interacts physically with liganded Egr-1 in the proximal promoter via a DNA-looping mechanism to mediate repression by c-Rel. In the absence of progesterone induction, RUSH-1beta replaces RUSH-1alpha binding, Egr-1 and c-Rel are unavailable as molecular ties, and DNA looping is disfavored.

Uteroglobin: a steroid-inducible immunomodulatory protein that founded the Secretoglobin superfamily.

Blastokinin or uteroglobin (UG) is a steroid-inducible, evolutionarily conserved, secreted protein that has been extensively studied from the standpoint of its structure and molecular biology. However, the physiological function(s) of UG still remains elusive. Isolated from the uterus of rabbits during early pregnancy, UG is the founding member of a growing superfamily of proteins called Secretoglobin (Scgb). Numerous studies demonstrated that UG is a multifunctional protein with antiinflammatory/ immunomodulatory properties. It inhibits soluble phospholipase A(2) activity and binds and perhaps sequesters hydrophobic ligands such as progesterone, retinols, polychlorinated biphenyls, phospholipids, and prostaglandins. In addition to its antiinflammatory activities, UG manifests antichemotactic, antiallergic, antitumorigenic, and embryonic growth-stimulatory activities. The tissue-specific expression of the UG gene is regulated by several steroid hormones, although a nonsteroid hormone, prolactin, further augments its expression in the uterus. The mucosal epithelia of virtually all organs that communicate with the external environment express UG, and it is present in the blood, urine, and other body fluids. Although the physiological functions of this protein are still under investigation, a single nucleotide polymorphism in the UG gene appears to be associated with several inflammatory/autoimmune diseases. Investigations with UG-knockout mice revealed that the absence of this protein leads to phenotypes that suggest its critical homeostatic role(s) against oxidative damage, inflammation, autoimmunity, and cancer. Recent studies on UG-binding proteins (receptors) provide further insight into the multifunctional nature of this protein. Based on its antiinflammatory and antiallergic properties, UG is a potential drug target.

Prolactin and growth hormone signaling.

Prolactin (PRL) and growth hormone (GH) act by way of their receptors as either hormones (systemically) or cytokines (locally). The Jak2/Stat5 pathway is the principal route by which PRL/GH activate target genes. The availability of knockout mice for each member of this signaling cascade has provided opportunities to understand their unique interactions. Jak2 is important in alternative signal transduction schema such as the MAP kinase and PI3K/Akt pathways. The putative Jak2/RUSH pathway is based on the fact that RUSH mediates the ability of PRL to augment progesterone-dependent gene transcription. New evidence shows that suppressors, regulators, and degraders control Jak2/Stat5. This review focuses on the most recent advances in the field of PRL/GH signal transduction.

Prolactin signals through RUSH/SMARCA3 in the absence of a physical association with Stat5a.

Jak/Stat-mediated prolactin signal transduction culminates in the sequence-selective binding of Stat5a. However, in the absence of Stat-binding sites, a RUSH-binding element mediates the prolactin signal in the rabbit uteroglobin promoter. Speculation about the existence of a Jak/RUSH pathway prompted this series of experiments to examine potential interactions between RUSH and Stat5a. Profiles of Jak/Stat pathway-specific genes by RT-PCR showed that mRNA for Jak2 and Stat5a is expressed in the endometrium of estrous, progesterone-treated, and 5-day pseudopregnant rabbits. Interspecies microarrays showed that transcripts for Stat5a were present at equal concentrations in the endometrium regardless of hormone treatment. The absence of a physical interaction between RUSH and individual Stat proteins bound to enhancer sites was demonstrated with transcription factor interaction arrays. These studies confirm that transmission of the prolactin signal through RUSH occurs in the absence of a physical association with Stat5a. Although a strong physical interaction between RUSH and Egr-1 was identified with the same arrays, no Egr-1 consensus sites were found in the region of the uteroglobin promoter (-175/-80) that contains the authentic RUSH site. Because the major transducer molecules (Jak2, Stat5a) are activated by tyrosine phosphorylation, Western analysis of immunoprecipitated samples, and gel shift assays were used to show that tyrosine phosphorylation is required for RUSH-DNA binding. The precise role for Jak2 in this process remains undefined. By comparison, serine-threonine-specific protein phosphorylation had no effect on RUSH-DNA binding.

An Sp1-NF-Y/progesterone receptor DNA binding-dependent mechanism regulates progesterone-induced transcriptional activation of the rabbit RUSH/SMARCA3 gene.

Steroids regulate alternative splicing of rabbit RUSH/SMARCA3, an SWI/SNF-related transcription factor. Transactivation was evaluated in 2057 bp of genomic sequence. Truncation analysis identified a minimal 252-bp region with strong basal promoter activity in transient transfection assays. The size of the 5'-untranslated region (233 bp) and the transcription start site were determined by primer extension analysis. The transcription start site mapped to a consensus initiator (Inr) element in a TATA-less region with a downstream promoter element (+29). These elements were authenticated by mutation/deletion analysis. The Inr/downstream promoter element combination is conserved in the putative core promoter (-35/+35) of the human ortholog, suggesting that transcription initiation is similarly conserved. Two Sp1 sites that are also conserved in the putative promoter of human SMARCA3 and a RUSH binding site (-616/-611) that is unique to the rabbit promoter repress basal transcription. These sites were variously authenticated by gel shift and chromatin immunoprecipitation assays. Analysis of the proximal promoter showed the -162/+90 region was required for progesterone responsiveness in transient transfection assays. Subsequent mutation/deletion analysis revealed a progesterone receptor half-site mediated induction by progesterone. An overlapping Y-box (in the reverse ATTGG orientation) repressed basal transcription and progesterone-induced transcriptional activation in the presence of the Sp1 sites. The specificity of progesterone receptor and transcription factor NF-Y binding were authenticated by gel shift assays. Chromatin immunoprecipitation assays confirmed the Y-box effects were mediated in a DNA binding-dependent fashion. This represents a unique regulatory scenario in which ligand-dependent transactivation by the progesterone receptor is subject to Sp1/NF-Y repression.

Identification of the RUSH consensus-binding site by cyclic amplification and selection of targets: demonstration that RUSH mediates the ability of prolactin to augment progesterone-dependent gene expression.

RUSH-1alpha(beta) transcription factors were cloned by recognition site screening with an 85-bp region (-170/-85) of the rabbit uteroglobin gene. Deletion analysis showed this region was essential to prolactin (PRL) action, but conclusions were limited by the complexity of the large deletion. Cyclic amplification and selection of targets (CASTing) was used to identify the RUSH-binding site (-126/-121). Endometrial nuclear proteins were incubated with a pool of degenerate oligonucleotides and immunoprecipitated with RUSH-1alpha(beta) antibodies. Bound DNA was amplified by PCR. The consensus motif (MCWTDK) was identified after five rounds of CASTing, authenticated by CASTing with RUSH-1alpha-specific antibodies and recombinant protein, and refined with EMSA. Dissociation rate constants (K(d) = 0.1-1.0 nM; r = 0.99) revealed high-affinity binding. Chromatin immunoprecipitation confirmed in vivo binding of RUSH to the transcriptionally active uteroglobin promoter. CASTing also revealed RUSH-GATA transcription factor interactions. Endometrial GATA-4 expression is progesterone dependent (Northern analysis) and preferentially localized in the epithelium (in situ hybridization). Although physically affiliated with RUSH, uterine forms of GATA-4 were not required for RUSH-DNA binding. Site-directed mutagenesis and transient transfection assays showed the RUSH motif mediates the ability of PRL to augment progesterone-dependent uteroglobin transcription. RUSH is central to the mechanism whereby PRL augments progesterone-dependent gene transcription.

The reproductive importance of P-type ATPases.

P-type ATPases are integral membrane proteins that use the free energy of ATP hydrolysis to generate transmembrane electrochemical ion gradients to support a variety of cellular processes. They have eight signature motifs, eight or ten transmembrane domains, highly conserved phosphorylation and ATP-binding sites, and similar hydropathic profiles. This review summarizes recent insights in the relationship of P-type ATPases to successful reproduction, and the hormone dependence of some family members. Because protein topology is central to understanding the pump action of this family of enzymes, this review also describes the dramatic change in the primary structure of one family member that may mediate transcription in the uterus.