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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 acidβ-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 acidβ-oxidation could potentially serve as an alternative energy source and survival mechanism for breast cancer cells in response to hypoxia-mediated VPS4B dysfunction.

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 alpha-subunit expression, little is known about the molecular mechanisms by which cells modulate alpha-ENaC gene expression. Previously, we have shown that salivary alpha-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 alpha-ENaC expression is regulated in salivary epithelial Pa-4 cells. A region located between -1.5 and -1.0 kilobase pairs of the alpha-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 identified alpha-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 alpha-ENaC expression through the identified GRE. In summary, this study presents a mechanism by which alpha-ENaC expression is regulated in salivary epithelial cells.

Proline-rich proteins are a family of proteins that exhibit unique features including an unusual high proline content and salivary-specificity. As a major constituent in the salivary secretion of higher primates, proline-rich proteins may have biological roles in oral lubrication and protection. In this article, the genomic structure and regulation by cAMP of one of the macaque salivary proline-rich protein genes, MnP4, is reviewed. The evolution of this multigene family of proteins is also discussed.

Krüppel-associated box (KRAB) domain-associated protein 1 [KAP1, also known as transcription intermediary factor-1beta (TIF1beta)] is a ubiquitous transcriptional co-repressor that is susceptible to phosphorylation at Ser(824) by ataxia-telangiectasia mutated (ATM) and to modification by small ubiquitin-like modifying (SUMO) proteins. Here, we found that, whereas the protein phosphatase 1alpha isoform (PP1alpha) directly interacted with KAP1 under basal conditions, PP1beta interacted with KAP1 only in response to genotoxic stress. Changes in the abundance of PP1alpha or PP1beta had differential effects on the phosphorylation and SUMOylation states of KAP1 under basal conditions and in response to DNA double-strand breaks (DSBs). Chromatin immunoprecipitation and re-immunoprecipitation experiments revealed that PP1alpha and PP1beta were recruited to KAP1 with different kinetics before and after the induction of DNA DSBs, which provided a mechanistic basis for the switch in the phosphorylation and SUMOylation states of KAP1. PP1beta-dependent SUMOylation of KAP1 occurred by mechanisms that were dependent and independent of the phosphorylation status of Ser(824). We posit a mechanism whereby the combined actions of PP1alpha and PP1beta cause dephosphorylation of KAP1 at Ser(824) and assure its SUMOylation to counter the effect of ATM, thereby regulating the transcription of KAP1 target genes in unstressed and stressed cells.

Previously, we reported a rat S1 protein that is antigenically related to statin, a nonproliferating cell-specific marker; however, it shares high homology with the known human elongation factor-1 alpha (EF-1 alpha). To differentiate S1 from rat EF-1 alpha and to study their respective regulation for expression, a rat EF-1 alpha cDNA clone was isolated and characterized. The nucleotide and deduced amino acid sequences of this partial rat EF-1 alpha cDNA are compared with that of human and mouse as well as with rat S1. Both their messages were detected in rat brain by EF-1 alpha- or S1-specific probes. However, the mRNA encoding EF-1 alpha is more abundant than that encoding S1. S1 and EF-1 alpha expression were investigated in the parotid and submandibular glands of untreated rats and those treated with isoproterenol, a proliferation-inducing catecholamine. Quantitative solution hybridization demonstrated a dramatic reduction (approximately 68 ) in the S1 mRNA following isoproterenol injection in proliferation-responsive parotid glands and a mild reduction (approximately 20 ) of S1 steady-state messages in the proliferation-refractile submandibular glands. A slight increase or no changes of EF-1 alpha levels in both parotid and submandibular glands following isoproterenol treatment are also observed. Therefore, the EF-1 alpha and S1 genes are different genes, both expressed and regulated in vivo, but in differential quantitative and qualitative patterns.

Previously, we have demonstrated that oxidative stress or Ras/ERK activation leads to the transcriptional repression of alpha-subunit of epithelial Na(+) channel (ENaC) in lung and salivary epithelial cells. Here, we further investigated the coordinated molecular mechanisms by which alpha-ENaC expression is regulated. Using both stable and transient transfection assays, we demonstrate that the overexpression of high mobility group protein I-C (HMGI-C), a Ras/ERK-inducible HMG-I family member, represses glucocorticoid receptor (GR)/dexamethasone (Dex)-stimulated alpha-ENaC/reporter activity in salivary epithelial cells. Northern analyses further confirm that the expression of endogenous alpha-ENaC gene in salivary Pa-4 cells is suppressed by an ectopic HMGI-C overexpression. Through yeast two-hybrid screening and co-immunoprecipitation assays from eukaryotic cells, we also discovered the interaction between HMGI-C and PIAS3 (protein inhibitor of activated STAT3 (signal transducer and activator of transcription 3)). A low level of ectopically expressed PIAS3 cooperatively inhibits GR/Dex-dependent alpha-ENaC transcription in the presence of HMGI-C. Reciprocally, HMGI-C expression also coordinately enhances PIAS3-mediated repression of STAT3-dependent transactivation. Moreover, overexpression of antisense HMGI-C construct is capable of reversing the repression mediated by Ras V12 on GR- and STAT3-dependent transcriptional activation. Together, our results demonstrate that Ras/ERK-mediated induction of HMGI-C is required to effectively repress GR/Dex-stimulated transcription of alpha-ENaC gene and STAT3-mediated transactivation. These findings delineate a network of inhibitory signaling pathways that converge on HMGI-C.PIAS3 complex, causally associating Ras/ERK activation with the repression of both GR and STAT3 signaling pathways in salivary epithelial cells.

Etk/Bmx is a member of the Tec family of cytoplasmic non-receptor tyrosine kinases known to express in epithelial cells. We demonstrate herein that Etk activation in stably Etk-transfected epithelial Pa-4 cells resulted in a consistently increased transepithelial resistance (TER). After 24 h of hypoxic (1% O2) exposure, the TER and equivalent active ion transport rate ( Ieq) were reduced to eqwere maintained at comparable and 60% levels, respectively, relative to their normoxic controls in cells with Etk activation. Moreover, Pa-4 cells exhibited an abundant actin stress fiber network with a diffuse distribution of β-catenin at the cell periphery. By contrast, Etk-activated cells displayed a redistribution of actin to an exclusively peripheral network, with a discrete band of β-catenin also concentrated at the cell periphery, and an altered occludin distribution profile. On the basis of these findings, we propose that Etk may be a novel regulator of epithelial junctions during physiological and pathophysiological conditions.

Previous studies have shown that an inducible Raf-1 kinase protein, DeltaRaf-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 DeltaRaf-1:ER activation. Through this approach, the gene encoding the alpha-subunit of the amiloride-sensitive epithelial sodium channel (alpha-ENaC) was identified as a target of activated Raf-1 kinases. alpha-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 alpha-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 alpha-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 alpha-ENaC-reporter and activated Raf expression construct(s), we provide the first evidence that activation of the ERK pathway down-regulates alpha-ENaC expression at the transcriptional level. Elucidating the molecular programming that modulates the expression of the alpha-subunit may provide new insights into the modulation of sodium reabsorption across epithelia.

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 (DeltaRaf-1:ER) and used mRNA differential display in search of messages induced by DeltaRaf-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-C expression in the Pa-4 cells. The induction of HMGI-C mRNA 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 the HMGI-C induction was highlighted by the result that the MAP kinase kinase (MEK) inhibitor, PD 98059, blocked DeltaRaf-1:ER- and 12-O-tetradecanoylphorbol-13-acetate-stimulated HMGI-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.

Proline-rich proteins (PRPs) are selectively expressed in the acinar cells of the salivary glands and are inducible by beta-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(Delta25-195), 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.