H. Helen Lin

Kinase activation of the non-receptor tyrosine kinase Etk/BMX alone is sufficient to transactivate STAT-mediated gene expression in salivary and lung epithelial cells.

Etk/BMX is a non-receptor protein tyrosine kinase that requires a functional phosphatidylinositol 3-kinase via the pleckstrin homology domain to be activated by cytokine. In the present study, a conditionally active form of Etk was constructed by fusing the hormone-binding domain of estrogen receptor (ER) to an amino terminus truncated form of Etk, PHΔ1–68Etk, to generate ΔEtk:ER. In stably transfected Pa-4ΔEtk:ER cells, the activity of ΔEtk:ER was stimulated within minutes by the treatment of ΔEtk:ER stimulant, estradiol, and sustained for greater than 24 h. A robust induction in the phosphorylation of signal transducers and activators of transcription (STAT) proteins, including STAT1, STAT3, and STAT5, was accompanied with ΔEtk:ER activation. Moreover, the conditionally activated Etk stimulated STAT1- and STAT5-dependent reporter activities by ∼160- and ∼15-fold, respectively, however, elicited only a modest STAT3-mediated reporter activation. Qualitatively comparable results were obtained in lung A549 cells, indicating that ΔEtk:ER inducible system could function in an analogous fashion in different epithelial cells. Furthermore, we demonstrated that Etk activation alone augmented cyclin D1 promoter/enhancer activity via its STAT5 response element in both Pa-4ΔEtk:ER and A549 cells. Altogether, these findings support the notion that the activation of Etk kinase is sufficient to transactivate STAT-mediated gene expression. Hence, our inducible ΔEtk:ER system represents a novel approach to investigate the biochemical events following Etk activation and to evaluate the contribution by kinase activation of Etk alone or in conjunction with other signaling pathway(s) to the ultimate biological responses.

THE AMILORIDE-SENSITIVE EPITHELIAL SODIUM CHANNEL ALPHA -SUBUNIT IS TRANSCRIPTIONALLY DOWN-REGULATED IN RAT PAROTID CELLS BY THE EXTRACELLULAR SIGNAL- REGULATED PROTEIN KINASE PATHWAY

Previous studies have shown that an inducible Raf-1 kinase protein, ΔRaf-1:ER, activates the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK)-signaling pathway, which is required for the transformation of the rat salivary epithelial cell line, Pa-4. Differential display polymerase chain reaction was employed to search for mRNAs repressed by ΔRaf-1:ER activation. Through this approach, the gene encoding the α-subunit of the amiloride-sensitive epithelial sodium channel (α-ENaC) was identified as a target of activated Raf-1 kinases. α-ENaC down-regulation could also be seen in cells treated with 12-O-tetradecanoyl-1-phorbol-13-acetate (TPA), indicating that the repression of steady-state α-ENaC mRNA level was dependent upon the activity of protein kinase C, the target of TPA, as well. Pretreatment of cells with a specific inhibitor of the ERK kinase pathway, PD 98059, markedly abolished the down-regulation of α-ENaC expression, consistent with the hypothesis that the ERK kinase-signaling pathway is involved in TPA-mediated repression. Moreover, through the use of transient transfection assays with α-ENaC-reporter and activated Raf expression construct(s), we provide the first evidence that activation of the ERK pathway down-regulates α-ENaC expression at the transcriptional level. Elucidating the molecular programming that modulates the expression of the α-subunit may provide new insights into the modulation of sodium reabsorption across epithelia.

High Mobility Group A2 Potentiates Genotoxic Stress in Part through the Modulation of Basal and DNA Damage-Dependent Phosphatidylinositol 3-Kinase-Related Protein Kinase Activation

The high mobility group A2 (HMGA2) protein belongs to the architectural transcription factor HMGA family, playing a role in chromosomal organization and transcriptional regulation. We and others have previously reported that ectopic HMGA2 expression is associated with neoplastic transformation and anchorage-independent cell proliferation. Here, we reported a correlation between increased HMGA2 expression and enhanced chemosensitivity towards topoisomerase II inhibitor, doxorubicin, in breast cancer cells. Using cells exhibiting differential HMGA2 expression and small interfering RNA technique, we showed that HMGA2 expression modulates cellular response to the genotoxicity of DNA double-strand breaks. Notably, HMGA2 enhances doxorubicin-elicited cell cycle delay in sub-G1 and G2-M and augments cell cycle dysregulation on cotreatment of doxorubicin and caffeine. We further reported that HMGA2 induces a persistent Ser139 phosphorylation of histone 2A variant X, analogous to the activation by doxorubicin-mediated genotoxic stress. Moreover, this HMGA2-dependent enhancement of cytotoxicity is further extended to other double-strand breaks elicited by cisplatin and X-ray irradiation and is not restricted to one cell type. Together, we postulated that the enhanced cytotoxicity by double-strand breaks in HMGA2-expressing cells is mediated, at least in part, through the signaling pathway of which the physiologic function is to maintain genome integrity. These findings should contribute to a greater understanding of the role of HMGA2 in promoting tumorigenesis and conveying (chemo)sensitivity towards doxorubicin and other related double-strand breaks.

PKCδ signaling: A dual role in regulating hypoxic stress-induced autophagy and apoptosis

The protein kinase C (PKC) family of serine/threonine kinases regulates diverse cellular function, including cell death, proliferation and survival. In particular, PKCδ governs the cellular homeostatic response against hypoxic stress. Autophagy, a lysosome-dependent degradative pathway, and apoptosis are two fundamental cellular pathways that respond to stress conditions, such as hypoxia, oxidative stress, and nutrient starvation. Recently, we uncovered a novel role for PKCδ in the early stage of hypoxic response where PKCδ activates autophagy by promoting JNK1-mediated Bcl-2 phosphorylation and dissociation of the Bcl-2/Beclin 1 complex. Whereas acute hypoxic stress promotes autophagy, we have previously reported that prolonged hypoxic stress caused the cleavage of PKCδ by caspase-3, resulting in the nuclear translocation of a constitutively active catalytic fragment of PKCδ, PKCδ-CF. Moreover, PKCδ-CF also serves a feed-forward function for the reciprocal PKCδ and caspase-3 proteolytic activation. Here, we discussed the requirement for PKCδ and JNK1 for hypoxia-induced autophagy, and the kinetic relationship among Bcl-2/Beclin 1 interaction, caspase-3 activation and the steady-state level of Beclin 1 during hypoxic exposure. Based on these results, we propose a model for understanding the PKCδ-dependent crosstalk mechanisms between autophagy and apoptosis, both induced by hypoxic stress. These findings collectively support a pivotal role for PKCδ in regulating hypoxic stress with hitherto unappreciated significance.

The Gene Expression of the Amiloride-sensitive Epithelial Sodium Channel α-Subunit Is Regulated by Antagonistic Effects between Glucocorticoid Hormone and Ras Pathways in Salivary Epithelial Cells

The functional expression of the amiloride-sensitive epithelial sodium channel (ENaC) in select epithelia is critical for maintaining electrolyte and fluid homeostasis. Although ENaC activity is strictly dependent upon its α-subunit expression, little is known about the molecular mechanisms by which cells modulate α-ENaC gene expression. Previously, we have shown that salivary α-ENaC expression is transcriptionally repressed by the activation of Raf/extracellular signal-regulated protein kinase pathway. Here, this work further investigates the molecular mechanism(s) by which α-ENaCexpression is regulated in salivary epithelial Pa-4 cells. A region located between −1.5 and −1.0 kilobase pairs of theα-ENaC 5′-flanking region is demonstrated to be indispensable for the maximal and Ras-repressible reporter expression. Deletional analyses using heterologous promoter constructs reveal that a DNA sequence between −1355 and −1269 base pairs functions as an enhancer conferring the high level of expression on reporter constructs, and this induction effect is inhibited by Ras pathway activation. Mutational analyses indicate that full induction and Ras-mediated repression require a glucocorticoid response element (GRE) located between −1323 and −1309 base pairs. The identifiedα-ENaC GRE encompassing sequence (−1334/−1306) is sufficient to confer glucocorticoid receptor/dexamethasone-dependent and Ras-repressible expression on both heterologous and homologous promoters. This report demon- strates for the first time that the cross-talk between glucocorticoid receptor and Ras/extracellular signal-regulated protein kinase signaling pathways results in an antagonistic effect at the transcriptional level to modulate α-ENaC expression through the identified GRE. In summary, this study presents a mechanism by which α-ENaC expression is regulated in salivary epithelial cells.

Oncogenic Raf-1 Induces the Expression of Non-histone Chromosomal Architectural Protein HMGI-C via a p44/p42 Mitogen-activated Protein Kinase-dependent Pathway in Salivary Epithelial Cells

The enzyme activity of mitogen-activated protein kinase (MAP kinase) increases in response to agents acting on a variety of cell surface receptors, including receptors linked to heterotrimeric G proteins. In this report, we demonstrated that Raf-1 protein kinase activity in the mouse parotid glands was induced by chronic isoproterenol administration in whole animals. To investigate the molecular nature underlying cellular responses to Raf-1 activation, we have stably transfected rat salivary epithelial Pa-4 cells with human Raf-1-estrogen receptor fusion gene (ΔRaf-1:ER) and used mRNA differential display in search of messages induced by ΔRaf-1:ER activation. Through this approach, the gene encoding non-histone chromosomal protein HMGI-C was identified as one of the target genes activated by oncogenic Raf-1 kinase. Activation of Raf-1 kinase resulted in a delayed and sustained increase of HMGI-Cexpression in the Pa-4 cells. The induction of HMGI-CmRNA level is sensitive to both the protein synthesis inhibitor cycloheximide and transcription inhibitor actinomycin D. The role of the extracellular signal-related kinase (ERK) signaling pathway in theHMGI-C induction was highlighted by the result that the MAP kinase kinase (MEK) inhibitor, PD 98059, blocked ΔRaf-1:ER- and 12-O-tetradecanoylphorbol-13-acetate-stimulatedHMGI-C induction. Altogether, these findings support the notion that the Raf/MEK/ERK signaling module, at least in part, regulates transcriptional activation of the chromosomal architectural protein HMGI-C.

Involvement of Nuclear Orphan Receptor NGFI-B in Transcriptional Activation of Salivary-specific R15 Gene by cAMP

Proline-rich proteins (PRPs) are selectively expressed in the acinar cells of the salivary glands and are inducible by β-agonist isoproterenol and dietary tannins. In the previous studies of rat PRP gene, R15, the 5′-flanking region up to −1.7 kilobase pairs (kb), which was thought to contain the necessary proximal regulatory elements, failed to confer the catecholamine isoproterenol and dietary tannin inducibility to the transgene expression in the salivary glands of transgenic mice. Here we analyzed distal 5′-flanking region of R15 in order to understand the mechanisms of tissue-specific and inducible gene regulation. An upstream regulatory region located between −2.4 and −1.7 kb of the R15 5′-flanking region is demonstrated to be indispensable for the salivary-specific and inducible reporter gene expression in vivo, by transgenic approach. Element(s) within the 0.7-kb (−2.4 to −1.7) region that is able to cis-activate the expression of a heterologous reporter gene expression is further elucidated by transient transfection assays in vitro. Three distinct nuclear orphan receptor NGFI-B regulatory sequences are identified within a 184-base pair (bp) minimal control region extended from −1995 to −1812 nucleotides relative to the transcription start site. When reporter gene containing this 184-bp control region and heterologous promoter was cotransfected with the NGFI-B expression construct, a transactivation that mimics the effect of cAMP is observed in the parotid cells. Finally, mutations on all three identified NGFI-B binding sites and coexpression of a dominant negative mutant construct, pCMV-NGFI-B(Δ25-195), abolish this transactivation mediated by NGFI-B. In summary, these data suggest that the inducible nuclear orphan receptor NGFI-B may participate in the regulation of salivary acinar cell-specific and inducible expression of the rat R15 gene via three distinct distal NGFI-B sites.

Regulation of salivary-gland-specific gene expression.

The results from in vivo transgenic and in vitro transfection studies designed to identify cis-element(s) and transfactor(s) governing the salivary proline-rich proteins (PRPs), amylase, and parotid secretory protein (PSP) gene expression are utilized as a paradigm to discuss the regulation of salivary-specific gene expression. Particular attention is given to the molecular mechanism(s) underlying the salivary PRP R15 gene regulation. In rodents, the PRPs are selectively expressed in the acinar cells of salivary glands, and are inducible by the beta-agonist isoproterenol and by dietary tannins. The results from a series of experiments using chimeric reporter constructs containing different lengths of the R15 distal enhancer region, their mutations, and various expressing constructs are analyzed and discussed. These data suggest that the inducible nuclear orphan receptor NGFI-B may participate in the regulation of salivary acinar-cell-specific and inducible expression of the rat R15 gene via three distinct distal NGFI-B sites. Taken together, a model for the induction of R15 gene expression by Ipr is proposed. However, the exact molecular basis of this NGFI-B-mediated transactivation of cAMP-regulated R15 expression remains to be established.

An Internal Standard-Assisted Synthesis and Degradation Proteomic Approach Reveals the Potential Linkage between VPS4B Depletion and Activation of Fatty Acid β-Oxidation in Breast Cancer Cells

The endosomal/lysosomal system, in particular the endosomal sorting complexes required for transport (ESCRTs), plays an essential role in regulating the trafficking and destination of endocytosed receptors and their associated signaling molecules. Recently, we have shown that dysfunction and down-regulation of vacuolar protein sorting 4B (VPS4B), an ESCRT-III associated protein, under hypoxic conditions can lead to the abnormal accumulation of epidermal growth factor receptor (EGFR) and aberrant EGFR signaling in breast cancer. However, the pathophysiological consequences of VPS4B dysfunction remain largely elusive. In this study, we used an internal standard-assisted synthesis and degradation mass spectrometry (iSDMS) method, which permits the direct measurement of protein synthesis, degradation and protein dynamic expression, to address the effects of VPS4B dysfunction in altering EGF-mediated protein expression. Our initial results indicate that VPS4B down-regulation decreases the expression of many proteins involved in glycolytic pathways, while increased the expression of proteins with roles in mitochondrial fatty acid β-oxidation were up-regulated in VPS4B-depleted cells. This observation is also consistent with our previous finding that hypoxia can induce VPS4B down-regulated, suggesting that the adoption of fatty acid β-oxidation could potentially serve as an alternative energy source and survival mechanism for breast cancer cells in response to hypoxia-mediated VPS4B dysfunction.

Autophagic reliance promotes metabolic reprogramming in oncogenic KRAS-driven tumorigenesis

ABSTRACT Defects in basal autophagy limit the nutrient supply from recycling of intracellular constituents. Despite our understanding of the prosurvival role of macroautophagy/autophagy, how nutrient deprivation, caused by compromised autophagy, affects oncogenic KRAS-driven tumor progression is poorly understood. Here, we demonstrate that conditional impairment of the autophagy gene Atg5 (atg5-KO) extends the survival of KRASG12V-driven tumor-bearing mice by 38%. atg5-KO tumors spread more slowly during late tumorigenesis, despite a faster onset. atg5-KO tumor cells displayed reduced mitochondrial function and increased mitochondrial fragmentation. Metabolite profiles indicated a deficiency in the nonessential amino acid asparagine despite a compensatory overexpression of ASNS (asparagine synthetase), key enzyme for de novo asparagine synthesis. Inhibition of either autophagy or ASNS reduced KRASG12V-driven tumor cell proliferation, migration, and invasion, which was rescued by asparagine supplementation or knockdown of MFF (mitochondrial fission factor). Finally, these observations were reflected in human cancer-derived data, linking ASNS overexpression with poor clinical outcome in multiple cancers. Together, our data document a widespread yet specific asparagine homeostasis control by autophagy and ASNS, highlighting the previously unrecognized role of autophagy in suppressing the metabolic barriers of low asparagine and excessive mitochondrial fragmentation to permit malignant KRAS-driven tumor progression.