Tryptophan Metabolites Target Transmembrane and Immunoglobulin Domain-Containing 1 Signaling to Augment Renal Tubular Injury.

Chronic kidney disease (CKD) is characterized by the accumulation of uremic toxins and renal tubular damage. Tryptophan-derived uremic toxins [indoxyl sulfate (IS) and kynurenine (Kyn)] are well-characterized tubulotoxins. Emerging evidence suggests that transmembrane and immunoglobulin domain-containing 1 (TMIGD1) protects tubular cells and promotes survival. However, the direct molecular mechanism(s) underlying how these two opposing pathways crosstalk remains unknown. We posited that IS and Kyn mediate tubular toxicity through TMIGD1 and the loss of TMIGD1 augments tubular injury. Results from the current study showed that IS and Kyn suppressed TMIGD1 transcription in tubular cells in a dose-dependent manner. The wild-type CCAAT enhancer-binding protein ß (C/EBPß) enhanced, whereas a dominant-negative C/EBPß suppressed, TMIGD1 promoter activity. IS down-regulated C/EBPß in primary human renal tubular cells. The adenine-induced CKD, unilateral ureteric obstruction, and deoxycorticosterone acetate salt unilateral nephrectomy models showed reduced TMIGD1 expression in the renal tubules, which correlated with C/EBPß expression. C/EBPß levels negatively correlated with the IS and Kyn levels. Inactivation of TMIGD1 in mice significantly lowered acetylated tubulin, decreased tubular cell proliferation, caused severe tubular damage, and worsened renal function. Thus, the current results demonstrate that TMIGD1 protects renal tubular cells from renal injury in different models of CKD and uncovers a novel mechanism of tubulotoxicity of tryptophan-based uremic toxins.

N-Glycosylation regulates ligand-dependent activation and signaling of vascular endothelial growth factor receptor 2 (VEGFR2).

The tumor microenvironment and proinflammatory signals significantly alter glycosylation of cell-surface proteins on endothelial cells. By altering the N-glycosylation machinery in the endoplasmic reticulum and Golgi, proinflammatory cytokines promote the modification of endothelial glycoproteins such as vascular endothelial growth factor receptor 2 (VEGFR2) with sialic acid-capped N-glycans. VEGFR2 is a highly N-glycosylated receptor tyrosine kinase involved in pro-angiogenic signaling in physiological and pathological contexts, including cancer. Here, using glycoside hydrolase and kinase assays and immunoprecipitation and MS-based analyses, we demonstrate that N-linked glycans at the Asn-247 site in VEGFR2 hinder VEGF ligand-mediated receptor activation and signaling in endothelial cells. We provide evidence that cell surface-associated VEGFR2 displays sialylated N-glycans at Asn-247 and, in contrast, that the nearby sites Asn-145 and Asn-160 contain lower levels of sialylated N-glycans and higher levels of high-mannose N-glycans, respectively. Furthermore, we report that VEGFR2 Asn-247-linked glycans capped with sialic acid oppose ligand-mediated VEGFR2 activation, whereas the uncapped asialo-glycans favor activation of this receptor. We propose that N-glycosylation, specifically the capping of N-glycans at Asn-247 by sialic acid, tunes ligand-dependent activation and signaling of VEGFR2 in endothelial cells.

The cell adhesion molecule IGPR-1 is activated by and regulates responses of endothelial cells to shear stress.

Vascular endothelial cells respond to blood flow-induced shear stress. However, the mechanisms through which endothelial cells transduce mechanical signals to cellular responses remain poorly understood. In this report, using tensile-force assays, immunofluorescence and atomic force microscopy, we demonstrate that immunoglobulin and proline-rich receptor-1 (IGPR-1) responds to mechanical stimulation and increases the stiffness of endothelial cells. We observed that IGPR-1 is activated by shear stress and tensile force and that flow shear stress-mediated IGPR-1 activation modulates remodeling of endothelial cells. We found that under static conditions, IGPR-1 is present at the cell-cell contacts; however, under shear stress, it redistributes along the cell borders into the flow direction. IGPR-1 activation stimulated actin stress fiber assembly and cross-linking with vinculin. Moreover, we noted that IGPR-1 stabilizes cell-cell junctions of endothelial cells as determined by staining of cells with ZO1. Mechanistically, shear stress stimulated activation of AKT Ser/Thr kinase 1 (AKT1), leading to phosphorylation of IGPR-1 at Ser-220. Inhibition of this phosphorylation prevented shear stress-induced actin fiber assembly and endothelial cell remodeling. Our findings indicate that IGPR-1 is an important player in endothelial cell mechanosensing, insights that have important implications for the pathogenesis of common maladies, including ischemic heart diseases and inflammation.

TMIGD1 acts as a tumor suppressor through regulation of p21Cip1/p27Kip1 in renal cancer.

Renal cell carcinoma (RCC) is a high-risk metastasizing tumor with a poor prognosis and poorly understood mechanism. In this study, we demonstrate that transmembrane and immunoglobulin domain-containing 1 (TMIGD1) is a novel tumor suppressor that is highly expressed in normal renal tubular epithelial cells, but it is downregulated in human renal cancer. We have identified CCAAT/enhancer-binding proteinß (C/EBPß, also called LAP) as a key transcriptional regulator of TMIGD1, whose loss of expression is responsible for downregulation of TMIGD1 in RCC. Transcriptionally active C/EBPß/LAP physically interacted with and increased TMIGD1 promoter activity and expression of TMIGD1. Re-introduction of TMIGD1 into renal tumor cells significantly inhibited tumor growth and metastatic behaviors such as morphogenic branching and cell migration. Restoring TMIGD1 expression in renal tumor cells stimulated phosphorylation of p38MAK, induced expression of p21CIP1 (cyclin-dependent kinase inhibitor 1), and p27KIP1 (cyclin-dependent kinase inhibitor 1B) expression, key cell cycle inhibitor proteins involved in regulation of the cell cycle. The present study identifies TMIGD1 as a novel candidate tumor suppressor gene and provides important insight into pathobiology of RCC that could lead to a better diagnosis and possible novel therapy for RCC.

MINAR1 is a Notch2-binding protein that inhibits angiogenesis and breast cancer growth.

Intrinsically disordered proteins (IDPs)/intrinsically unstructured proteins are characterized by the lack of fixed or stable tertiary structure, and are increasingly recognized as an important class of proteins with major roles in signal transduction and transcriptional regulation. In this study, we report the identification and functional characterization of a previously uncharacterized protein (UPF0258/KIAA1024), major intrinsically disordered Notch2-associated receptor 1 (MINAR1). While MINAR1 carries a single transmembrane domain and a short cytoplasmic domain, it has a large extracellular domain that shares no similarity with known protein sequences. Uncharacteristically, MINAR1 is a highly IDP with nearly 70% of its amino acids sequences unstructured. We demonstrate that MINAR1 physically interacts with Notch2 and its binding to Notch2 increases its stability and function. MINAR1 is widely expressed in various tissues including the epithelial cells of the breast and endothelial cells of blood vessels. MINAR1 plays a negative role in angiogenesis as it inhibits angiogenesis in cell culture and in mouse matrigel plug and zebrafish angiogenesis models. Furthermore, while MINAR1 is highly expressed in the normal human breast, its expression is significantly downregulated in advanced human breast cancer and its re-expression in breast cancer cells inhibited tumor growth. Our study demonstrates that MINAR1 is an IDP that negatively regulates angiogenesis and growth of breast cancer cells.

Site-Specific N-Glycosylation of Endothelial Cell Receptor Tyrosine Kinase VEGFR-2.

Vascular endothelial growth factor receptor-2 (VEGFR-2) is an important receptor tyrosine kinase (RTK) that plays critical roles in both physiologic and pathologic angiogenesis. The extracellular domain of VEGFR-2 is composed of seven immunoglobulin-like domains, each with multiple potential N-glycosylation sites (sequons). N-glycosylation plays a central role in RTK ligand binding, trafficking, and stability. However, despite its importance, the functional role of N-glycosylation of VEGFR-2 remains poorly understood. The objectives of the present study were to characterize N-glycosylation sites in VEGFR-2 via enzymatic release of the glycans and concomitant incorporation of 18O into formerly N-glycosylated sites followed by tandem mass spectrometry (MS/MS) analysis to determine N-glycosylation site occupancy and the site-specific N-glycan heterogeneity of VEGFR-2 glycopeptides. The data demonstrated that all seven VEGFR-2 immunoglobulin-like domains have at least one occupied N-glycosylation site. MS/MS analyses of glycopeptides and deamidated, deglycosylated (PNGase F-treated) peptides from ectopically expressed VEGFR-2 in porcine aortic endothelial (PAE) cells identified N-glycans at the majority of the 17 potential N-glycosylation sites on VEGFR-2 in a site-specific manner. The data presented here provide direct evidence for site-specific, heterogeneous N-glycosylation and N-glycosylation site occupancy on VEGFR-2. The study has important implications for the therapeutic targeting of VEGFR-2, ligand binding, trafficking, and signaling.

IGPR-1 Is Required for Endothelial Cell-Cell Adhesion and Barrier Function.

Endothelial cell (EC) barrier function plays a prevalent regulatory mechanism for the integrity and homeostasis of blood vessels and modulates angiogenesis and immune responses. Cell adhesion molecules (CAMs) play a central role in the barrier function of ECs. Although Ig-containing and proline-rich receptor-1(IGPR-1) was recently identified as a novel CAM expressed in ECs, the molecular mechanisms underlying the function of IGPR-1 in ECs remain uncharacterized. In this report, we investigated the role of IGPR-1 in EC barrier function and the molecular mechanism of its activation in ECs. We demonstrate that IGPR-1 is localized to endothelial adherens junctions and, through trans-homophilic dimerization, regulates endothelial cell-cell adhesion and barrier function. Trans-homophilic dimerization of IGPR-1 stimulates the phosphorylation of serine 220 (Ser220), which is required for IGPR-1 to regulate endothelial barrier function and angiogenesis. Moreover, IGPR-1 chimera, which mimics the trans-homophilic dimerization of IGPR-1, induced a sustained phosphorylation of Ser220 upon stimulation with a ligand. Coordinated dimerization of IGPR-1 and its homophilic interaction modulates its adhesive function and Ser220 phosphorylation. This adhesive function of IGPR-1 contributes to the barrier function of ECs.

c-Cbl mediates the degradation of tumorigenic nuclear ß-catenin contributing to the heterogeneity in Wnt activity in colorectal tumors.

Despite the loss of Adenomatous Polyposis Coli (APC) in a majority of colorectal cancers (CRC), not all CRCs bear hallmarks of Wnt activation, such as nuclear ß-catenin. This underscores the presence of other Wnt regulators that are important to define, given the pathogenic and prognostic roles of nuclear ß-catenin in human CRC. Herein, we investigated the effect of Casitas B-lineage lymphoma (c-Cbl) on nuclear ß-catenin, which is an oncoprotein upregulated in CRC due to loss-of-function APC or gain-of-function CTNNB1 mutations. Despite mechanistic rationale and recent discoveries of c-Cbl's mutations in solid tumors, little is known about its functional importance in CRC. Our study in a cohort of human CRC patients demonstrated an inverse correlation between nuclear ß-catenin and c-Cbl. Further investigation showed that the loss of c-Cbl activity significantly enhanced nuclear ß-catenin and CRC tumor growth in cell culture and a mouse xenograft model. c-Cbl interacted with and downregulated ß-catenin in a manner that was independent of CTNNB1 or APC mutation status. This study demonstrates a previously unrecognized function of c-Cbl as a negative regulator of CRC.

RNF121 Inhibits Angiogenic Growth Factor Signaling by Restricting Cell Surface Expression of VEGFR-2.

Ligand stimulation promotes downregulation of RTKs, a mechanism by which RTKs, through the ubiquitination pathway are removed from the cell surface, causing a temporary termination of RTK signaling. The molecular mechanisms governing RTK trafficking and maturation in the endoplasmic reticulum (ER)/Golgi compartments are poorly understood. Vascular endothelial growth factor receptor-2 (VEGFR-2) is a prototypic RTK that plays a critical role in physiologic and pathologic angiogenesis. Here we demonstrate that Ring Finger Protein 121 (RNF121), an ER ubiquitin E3 ligase, is expressed in endothelial cells and regulates maturation of VEGFR-2. RNF121 recognizes newly synthesized VEGFR-2 in the ER and controls its trafficking and maturation. Over-expression of RNF121 promoted ubiquitination of VEGFR-2, inhibited its maturation and resulted a significantly reduced VEGFR-2 presence at the cell surface. Conversely, the shRNA-mediated knockdown of RNF121 in primary endothelial cells reduced VEGFR-2 ubiquitination and increased its cell surface level. The RING Finger domain of RNF121 is required for its activity toward VEGFR-2, as its deletion significantly reduced the effect of RNF121 on VEGFR-2. Additionally, RNF121 inhibited VEGF-induced endothelial cell proliferation and angiogenesis. Taken together, these data identify RNF121 as a key determinant of angiogenic signaling that restricts VEGFR-2 cell surface presence and its angiogenic signaling.

TMIGD1 is a novel adhesion molecule that protects epithelial cells from oxidative cell injury.

Oxidative damage to renal tubular epithelial cells is a fundamental pathogenic mechanism implicated in both acute kidney injury and chronic kidney diseases. Because epithelial cell survival influences the outcome of acute kidney injury and chronic kidney diseases, identifying its molecular regulators could provide new insight into pathobiology and possible new therapeutic strategies for these diseases. We have identified transmembrane and immunoglobulin domain-containing 1 (TMIGD1) as a novel adhesion molecule, which is highly conserved in humans and other species. TMIGD1 is expressed in renal tubular epithelial cells and promotes cell survival. The extracellular domain of TMIGD1 contains two putative immunoglobulin domains and mediates self-dimerization. Our data suggest that TMIGD1 regulates transepithelial electric resistance and permeability of renal epithelial cells. TMIGD1 controls cell migration, cell morphology, and protects renal epithelial cells from oxidative- and nutrient-deprivation-induced cell injury. Hydrogen peroxide-induced oxidative cell injury downregulates TMIGD1 expression and targets it for ubiquitination. Moreover, TMIGD1 expression is significantly affected in both acute kidney injury and in deoxy-corticosterone acetate and sodium chloride (deoxy-corticosterone acetate salt)-induced chronic hypertensive kidney disease mouse models. Taken together, we have identified TMIGD1 as a novel cell adhesion molecule expressed in kidney epithelial cells that protects kidney epithelial cells from oxidative cell injury to promote cell survival.

The novel function of JADE1S in cytokinesis of epithelial cells.

JADE1 belongs to a small family of PHD zinc finger proteins that interacts with histone acetyl transferase (HAT) HBO1 and is associated with chromatin. We recently reported JADE1 chromatin shuttling and phosphorylation during G2/M to G1 transition, which was sensitive to Aurora A inhibition. In the current study we examined mechanisms of the cell cycle regulation by the small isoform of JADE1 protein, JADE1S, and report data showing that JADE1S has a novel function in the regulation of cytokinesis. Using FACS assays, we show that, JADE1S depletion facilitated rates of G1-cells accumulation in synchronously dividing HeLa cell cultures. Depletion of JADE1S protein in asynchronously dividing cells decreased the proportion of cytokinetic cells, and increased the proportion of multi-nuclear cells, indicative of premature and failed cytokinesis. In contrast, moderate overexpression of JADE1S increased the number of cytokinetic cells in time- and dose- dependent manner, indicating cytokinetic delay. Pharmacological inhibition of Aurora B kinase resulted in the release of JADE1S-mediated cytokinetic delay and allowed progression of abscission in cells over-expressing JADE1S. Finally, we show that JADE1S protein localized to centrosomes in interphase and mitotic cells, while during cytokinesis JADE1S localized to the midbody. Neither JADE1L nor partner of JADE1, HAT HBO1 was localized to the centrosomes or midbodies. Our study identifies the novel role for JADE1S in regulation of cytokinesis and suggests function in Aurora B kinase-mediated cytokinesis checkpoint.

Hypoxia-induced expression of phosducin-like 3 regulates expression of VEGFR-2 and promotes angiogenesis.

Expression and activation of vascular endothelial growth factor receptor 2 (VEGFR-2) by VEGF ligands are the main events in the stimulation of pathological angiogenesis. VEGFR-2 expression is generally low in the healthy adult blood vessels, but its expression is markedly increased in the pathological angiogenesis. In this report, we demonstrate that phosducin-like 3 (PDCL3), a recently identified chaperone protein involved in the regulation of VEGFR-2 expression, is required for angiogenesis in zebrafish and mouse. PDCL3 undergoes N-terminal methionine acetylation, and this modification affects PDCL3 expression and its interaction with VEGFR-2. Expression of PDCL3 is regulated by hypoxia, the known stimulator of angiogenesis. The mutant PDCL3 that is unable to undergo N-terminal methionine acetylation was refractory to the effect of hypoxia. The siRNA-mediated silencing of PDCL3 decreased VEGFR-2 expression resulting in a decrease in VEGF-induced VEGFR-2 phosphorylation, whereas PDCL3 over-expression increased VEGFR-2 protein. Furthermore, we show that PDCL3 protects VEGFR-2 from misfolding and aggregation. The data provide new insights for the chaperone function of PDCL3 in angiogenesis and the roles of hypoxia and N-terminal methionine acetylation in PDCL3 expression and its effect on VEGFR-2.

Lysine methylation promotes VEGFR-2 activation and angiogenesis.

Activation of vascular endothelial growth factor receptor-2 (VEGFR-2), an endothelial cell receptor tyrosine kinase, promotes tumor angiogenesis and ocular neovascularization. We report the methylation of VEGFR-2 at multiple Lys and Arg residues, including Lys(1041), a residue that is proximal to the activation loop of the kinase domain. Methylation of VEGFR-2 was independent of ligand binding and was not regulated by ligand stimulation. Methylation of Lys(1041) enhanced tyrosine phosphorylation and kinase activity in response to ligands. Additionally, interfering with the methylation of VEGFR-2 by pharmacological inhibition or by site-directed mutagenesis revealed that methylation of Lys(1041) was required for VEGFR-2-mediated angiogenesis in zebrafish and tumor growth in mice. We propose that methylation of Lys(1041) promotes the activation of VEGFR-2 and that similar posttranslational modification could also regulate the activity of other receptor tyrosine kinases.

Identification of PDCL3 as a novel chaperone protein involved in the generation of functional VEGF receptor 2.

Angiogenesis, a hallmark step in tumor metastasis and ocular neovascularization, is driven primarily by the function of VEGF ligand on one of its receptors, VEGF receptor 2 (VEGFR-2). Central to the proliferation and ensuing angiogenesis of endothelial cells, the abundance of VEGFR-2 on the surface of endothelial cells is essential for VEGF to recognize and activate VEGFR-2. We have identified phosducin-like 3 (PDCL3, also known as PhLP2A), through a yeast two-hybrid system, as a novel protein involved in the stabilization of VEGFR-2 by serving as a chaperone. PDCL3 binds to the juxtamembrane domain of VEGFR-2 and controls the abundance of VEGFR-2 by inhibiting its ubiquitination and degradation. PDCL3 increases VEGF-induced tyrosine phosphorylation and is required for VEGFR-2-dependent endothelial capillary tube formation and proliferation. Taken together, our data provide strong evidence for the role of PDCL3 in angiogenesis and establishes the molecular mechanism by which it regulates VEGFR-2 expression and function.

Identification of IGPR-1 as a novel adhesion molecule involved in angiogenesis.

Angiogenesis-the growth of new blood vessels from preexisting vessels-is an important physiological process and is considered to play a key role in tumor growth and metastasis. We identified the immunoglobulin-containing and proline-rich receptor-1 (IGPR-1, also called TMIGD2) gene as a novel cell adhesion receptor that is expressed in various human organs and tissues, mainly in cells with epithelium and endothelium origins. IGPR-1 regulates cellular morphology, homophilic cell aggregation, and cell-cell interaction. IGPR-1 activity also modulates actin stress fiber formation and focal adhesion and reduces cell migration. Silencing of expression of IGPR-1 by small interfering RNA (siRNA) and by ectopic overexpression in endothelial cells showed that IGPR-1 regulates capillary tube formation in vitro, and B16F melanoma cells engineered to express IGPR-1 displayed extensive angiogenesis in the mouse Matrigel angiogenesis model. Moreover, IGPR-1, through its proline-rich cytoplasmic domain, associates with multiple Src homology 3 (SH3)-containing signaling proteins, including SH3 protein interacting with Nck (SPIN90/WISH), bullous pemphigoid antigen-1, and calcium channel ß2. Silencing of expression of SPIN90/WISH by siRNA in endothelial cells showed that SPIN90/WISH is required for capillary tube formation. These features of IGPR-1 suggest that IGPR-1 is a novel receptor that plays an important role in cell-cell interaction, cell migration, and angiogenesis.

PEST motif serine and tyrosine phosphorylation controls vascular endothelial growth factor receptor 2 stability and downregulation.

The internalization and degradation of vascular endothelial growth factor receptor 2 (VEGFR-2), a potent angiogenic receptor tyrosine kinase, is a central mechanism for the regulation of the coordinated action of VEGF in angiogenesis. Here, we show that VEGFR-2 is ubiquitinated in response to VEGF, and Lys 48-linked polyubiquitination controls its degradation via the 26S proteosome. The degradation and ubiquitination of VEGFR-2 is controlled by its PEST domain, and the phosphorylation of Ser1188/Ser1191 is required for the ubiquitination of VEGFR-2. F-box-containing ß-Trcp1 ubiquitin E3 ligase is recruited to S1188/S1191 VEGFR-2 and mediates the ubiquitination and degradation of VEGFR-2. The PEST domain also controls the activation of p38 mitogen-activated protein kinase (MAPK) through phospho-Y1173. The activation of p38 stabilizes VEGFR-2, and its inactivation accelerates VEGFR-2 downregulation. The VEGFR-2-mediated activation of p38 is established through the protein kinase A (PKA)/MKK6 pathway. PKA is recruited to VEGFR-2 through AKAP1/AKAP149, and its phosphorylation requires Y1173 of VEGFR-2. The study has identified a unique mechanism in which VEGFR-2 stability and degradation is modulated. The PEST domain acts as a dual modulator of VEGFR-2; the phosphorylation of S1188/S1191 controls ubiquitination and degradation via ß-Trcp1, where the phosphorylation of Y1173 through PKA/p38 MAPK controls the stability of VEGFR-2.

Role of c-Cbl-dependent regulation of phospholipase Cgamma1 activation in experimental choroidal neovascularization.

PURPOSE: Activation of phospholipase Cγ1 (PLCγ1) by vascular endothelial growth factor receptor (VEGFR)-2 is necessary for proliferation and tube formation of endothelial cells in vitro. Previous work has demonstrated that Casitas B-lineage lymphoma (c-Cbl) promotes ubiquitination of PLCγ1 and suppression of its tyrosine phosphorylation. This study was designed to evaluate the importance of PLCγ1 and c-Cbl in experimental choroidal neovascularization (CNV). METHODS: The role of PLCγ1 was studied in three models of angiogenesis: the endothelial cell culture system, the chorioallantoic membrane (CAM) assay, and the laser-induced CNV model. Endothelial cells were analyzed for the role of PLCγ1 in promoting tube formation. CAMs were incubated with pharmacologic agents that either inhibit or stimulate PLCγ1. CNV was induced in wild-type and c-Cbl-knockout mice, and the progression of CNV was evaluated by fluorescein angiography. RESULTS: Activation of PLCγ1 was necessary for tube formation of endothelial cells. PLCγ1 stimulation increased the growth of blood vessels and conversely, PLCγ1 inhibition decreased the growth of blood vessels in the CAM model. CNV lesions in the c-Cbl-knockout mice were significantly greater in number, more confluent, and increased in size with time, compared with those in the control wild-type mice. CONCLUSIONS: The data show that PLCγ1 plays an important role in angiogenesis. Loss of c-Cbl results in enhanced CNV in the eye. The study also shows that c-Cbl plays an important role in ocular angiogenesis, suggesting that modulation of c-Cbl activity or inhibition of PLCγ1 would be a compelling target for antiangiogenesis therapy.

Intrinsic sex differences in the early growth hormone responsiveness of sex-specific genes in mouse liver.

Sex differences in liver gene expression are dictated by sex differences in circulating GH profiles. Presently, the pituitary hormone dependence of mouse liver gene expression was investigated on a global scale to discover sex-specific early GH response genes that could contribute to sex-specific regulation of downstream GH targets and to ascertain whether intrinsic sex differences characterize hepatic responses to plasma GH stimulation. Global RNA expression analysis identified two distinct classes of sex-specific mouse liver genes: genes subject to positive regulation (class I) and genes subject to negative regulation by pituitary hormones (class II). Genes activated or repressed in hypophysectomized (Hypox) mouse liver within 30-90 min of GH pulse treatment at a physiological dose were identified as putative direct targets of GH action (early response genes). Intrinsic sex differences in the GH responsiveness of a subset of these early response genes were observed. Notably, 45 male-specific genes, including five encoding transcriptional regulators that may mediate downstream sex-specific transcriptional responses, were induced by GH within 30 min in Hypox male but not Hypox female mouse liver. The early GH response genes were enriched in 29 male-specific targets of the transcription factor myocyte enhancer factor 2, whose activation in hepatic stellate cells is associated with liver fibrosis leading to hepatocellular carcinoma, a male-predominant disease. Thus, the rapid activation by GH pulses of certain sex-specific genes is modulated by intrinsic sex-specific factors, which may be associated with prior hormone exposure (epigenetic mechanisms) or genetic factors that are pituitary-independent, and could contribute to sex differences in predisposition to liver cancer or other hepatic patho-physiologies.

Male-specific hepatic Bcl6: growth hormone-induced block of transcription elongation in females and binding to target genes inversely coordinated with STAT5.

The transcriptional repressor Bcl6 is a male-specific rat liver gene product and one of 24 early GH-response genes encoding DNA-binding proteins. Presently, the sex specificity of Bcl6 was shown to emerge at puberty, when hepatic Bcl6 mRNA was induced in males and repressed in females by the female plasma GH profile. Hepatic Bcl6 mRNA was increased to near-normal male levels in hypophysectomized females and was extinguished in intact males given a continuous GH infusion (female-like GH pattern). Bcl6 was also repressed in adult male somatostatin-deficient mice, where plasma GH profiles are female like. Hepatic Bcl6 RNA was rapidly down-regulated by GH pulse treatment, both in hypophysectomized male rats and in primary rat hepatocytes. Bcl6 was substantially induced in female mice deficient in hepatic signal transducer and activator of transcription (STAT)5a/STAT5b, suggesting that these STAT transcriptional mediators of GH signaling repress Bcl6. Indeed, STAT5 was bound to Bcl6 STAT5-binding region-B, previously associated with Bcl6 repression, in both male and female liver chromatin. STAT5 also bound to Bcl6 region-A in male chromatin but only during a plasma GH pulse. Analysis of primary transcripts (heterogeneous nuclear RNA) across the Bcl6 gene revealed a novel mechanism of GH-dependent sex specificity, with two apparent blocks in Bcl6 transcription elongation seen in female liver and in continuous GH-treated male liver, one early in intron 4 and one in exon 5, which together reduced transcription beyond exon 5 more than 300-fold. Finally, Bcl6 was bound to a subset of STAT5-binding sites in male liver chromatin, including a Socs2 STAT5-binding site where Bcl6 binding increased substantially between plasma GH pulses, i.e. when STAT5 binding was low. Bcl6 and STAT5 binding are thus inversely coordinated by the endogenous pulses of pituitary GH release, suggesting this male-specific transcriptional repressor modulates hepatic GH signaling to select STAT5 target genes.

Identification of ligand-induced proteolytic cleavage and ectodomain shedding of VEGFR-1/FLT1 in leukemic cancer cells.

Vascular endothelial growth factor receptor-1/fms-related tyrosine kinase 1 (VEGFR-1/FLT1) is expressed as a membrane-bound receptor tyrosine kinase and as an alternatively spliced soluble protein (sVEGFR-1) containing the 1-6 IgG-like domain of its ectodomain. sVEGFR-1 is known as a naturally occurring inhibitor of angiogenesis and as a surrogate marker for cancer progression; it is also linked to pregnancy-induced hypertension called preeclampsia and to avascularity of normal cornea. It remains an open question whether alternative mRNA splicing is the only mechanism by which sVEGFR-1 is generated. In this study, we show that in leukemic cancer cells, PlGF and VEGF-A both induce tyrosine phosphorylation of VEGFR-1 and render it susceptible to ectodomain shedding, resulting in the generation of sVEGFR-1 and an intracellular cytoplasmic fragment. Activation of protein kinase C and tumor necrosis factor-alpha-converting enzyme family metalloproteases are critically required for the occurrence of sVEGFR-1. Following the removal of the ectodomain, the remnant of VEGFR-1 remains attached to the membrane, and the activity of gamma-secretase/presenilin is required for its release from the cell membrane. We propose that sVEGFR-1 produced via ectodomain shedding plays a prominent role in the VEGF receptor system by antagonizing VEGF receptor signaling by acting as a dominant-negative form and/or forming a nonsignaling dimerizing complex with VEGF receptors.