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1.  Unfavorable Prognosis of CRTC1-MAML2 Positive Mucoepidermoid Tumors with CDKN2A Deletions 
Genes, chromosomes & cancer  2010;49(1):59-69.
The CRTC1-MAML2 fusion oncogene underlies the etiology of mucoepidermoid salivary gland carcinoma (MEC) where it confers a favorable survival outcome as compared with fusion-negative MEC. While these analyses suggested that detection of CRTC1-MAML2 serves as a useful prognostic biomarker, we recently identified outlier cases of fusion-positive MEC associated with advanced-staged lethal disease. To identify additional genetic alterations that might cooperate with CRTC1-MAML2 to promote disease progression, we performed a pilot high-resolution oligonucleotide array CGH (aCGH) and PCR-based genotyping study on 23 MEC samples including14 fusion-positive samples for which we had clinical outcome information. Unbiased aCGH analysis identified inactivating deletions within CDKN2A as a candidate poor prognostic marker which was confirmed by PCR-based analysis (CDKN2A deletions in 5/5 unfavorable fusion-positive cases and 0/9 favorable fusion-positive cases). We did not detect either activating EGFR mutations, nor copy number gains at the EGFR or ERBB2 loci as poor prognostic features for fusion-positive MEC in any of the tumor specimens. Prospective studies with larger case series will be needed to confirm that combined CRTC1-MAML2 and CDKN2A genotyping will optimally stage this disease.
PMCID: PMC2783528  PMID: 19827123
2.  Clock and light regulation of the CREB co-activator CRTC1 in the suprachiasmatic circadian clock 
The CREB/CRE transcriptional pathway has been implicated in circadian clock timing and light-evoked clock resetting. To date, much of the work on CREB in circadian physiology has focused on how changes in the phosphorylation state of CREB regulate the timing processes. However, beyond changes in phosphorylation, CREB-dependent transcription can also be regulated by the CREB co-activator CRTC (CREB-regulated transcription coactivator), also known as TORC (Transducer of Regulated CREB). Here we profiled both the rhythmic and light-evoked regulation of CRTC1 and CRTC2 in the murine suprachiasmatic nucleus (SCN), the locus of the master mammalian clock. Immunohistochemical analysis revealed rhythmic expression of CRTC1 in the SCN. CRTC1 expression was detected throughout the dorso-ventral extent of the SCN in the middle of the subjective day, with limited expression during early night, and late night expression levels intermediate between mid-day and early night levels. In contrast to CRTC1, robust expression of CRTC2 was detected during both the subjective day and night. During early and late subjective night, a brief light pulse induced strong nuclear accumulation of CRTC1 in the SCN. In contrast with CRTC1, photic stimulation did not affect the subcellular localization of CRTC2 in the SCN. Additionally, period1 reporter gene profiling and ChIP analysis revealed that CRTC1 was associated with CREB in the 5′ regulatory region of the period1 gene, and that over-expression of CRTC1 leads to a marked upregulation in period1 transcription. Together these data raise the prospect that CRTC1 plays a role in fundamental aspects of SCN clock timing and entrainment.
PMCID: PMC3745023  PMID: 23699513
3.  Adiponectin and thiazolidinedione targets CRTC2 to regulate hepatic gluconeogenesis 
Experimental & Molecular Medicine  2009;41(8):577-583.
During fasting periods, hepatic glucose production is enhanced by glucagon to provide fuels for other organs. This process is mediated via cAMP-dependent induction of the CREB regulated transcriptional coactivator (CRTC) 2, a critical transcriptional activator for hepatic gluconeogenesis. We have previously shown that CRTC2 activity is regulated by AMP activated protein kinase (AMPK) family members. Here we show that adiponectin and thiazolidinedione directly regulate AMPK to modulate CRTC2 activity in hepatocytes. Adiponectin or thiazolidinedione lowered glucose production from primary hepatocytes. Treatment of both reagents reduced gluconeogenic gene expression as well as cAMP-mediated induction of CRE reporter, suggesting that these reagents directly affect CREB/CRTC2- dependent transcription. Furthermore, adiponectin or thiazolidinedione mediated repression of CRE activity is largely blunted by co-expression of phosphorylation defective mutant CRTC2, underscoring the importance of serine 171 residue of this factor. Taken together, we propose that adiponectin and thiazolidinedione promote the modulation of AMPK-dependent CRTC2 activity to influence hepatic gluconeogenesis.
PMCID: PMC2739897  PMID: 19381067
adiponectin; AMP-activated protein kinases; CRTC2 protein, human; gluconeogenesis; liver; thiazolidinediones
4.  Developmental expression of survivin during embryonic submandibular salivary gland development 
The regulation of programmed cell death is critical to developmental homeostasis and normal morphogenesis of embryonic tissues. Survivin, a member of the inhibitors of apoptosis protein (IAP) family primarily expressed in embryonic cells, is both an anti-apoptosis and a pro-survival factor. Since our previous studies have demonstrated the importance of apoptosis during embryonic submandibular salivary gland (SMG) development, we postulated that survivin is a likely mediator of SMG epithelial cell survival.
We investigated the developmental expression of survivin in Pseudoglandular (~ E14), Canalicular (~ E15) and Terminal Bud (~ E17) Stage SMGs. We report a significant 26% increase in transcript levels between the Canalicular and Terminal Bud Stages. Immunohistochemical studies demonstrate nuclear-localized survivin protein in epithelial cells bounding forming lumina in Canalicular and Terminal Bud Stage SMGs.
Survivin is known to be a pro-survival and anti-apoptotic factor. Given that survivin translocation into the nucleus is required for the induction of entry into the cell cycle and the inhibition of apoptosis, our demonstration of nuclear-localized survivin protein in presumptive ductal and proacinar lumen-bounding cells suggests that survivin may be a key mediator of embryonic SMG epithelial cell survival.
PMCID: PMC31339  PMID: 11305929
5.  CRTC2 enhances HBV transcription and replication by inducing PGC1α expression 
Virology Journal  2014;11:30.
Hepatitis B virus (HBV) transcription and replication are essentially restricted to hepatocytes. Based on the HBV enhancer and promoter complex that links hepatic glucose metabolism to its transcription and replication, HBV adopts a regulatory system that is unique to the hepatic gluconeogenic genes. CRTC2, the CREB-regulated transcription coactivator 2, is a critical switch modulating the gluconeogenic program in response to both hormonal and intracellular signals. However, the relationship between CRTC2 and HBV transcription and replication remains unclear.
To analyze the influence of CRTC2 on HBV transcription and replication, CRTC2 expression construct or siRNA was cotransfected with plasmids containing enhancer II/core promoter complex-controlled luciferase or 1.3× wtHBV genome in Huh-7 cells. Luciferase activity, HBV core protein expression, HBV transcripts, and DNA replication intermediates were measured by luciferase assays, western blots, real-time polymerase chain reaction (PCR), and Southern blots, respectively. Forskolin (FSK) or phosphorylation-defective CRTC2 mutants were further utilized to elucidate the potential mechanism. siRNA against peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α) was also used to examine the mediator involved in CRTC2-regulated HBV biosynthesis in Huh-7 cells.
CRTC2 overexpression increased HBV transcription and replication in Huh-7 cells, including levels of core protein expression, mRNA, and DNA replication intermediates. Correspondingly, CRTC2 knock down by siRNA reduced HBV biosynthesis. FSK treatment strongly enhanced the effect of CRTC2 through triggering the dephosphorylation and nuclear entry of CRTC2. The phosphorylation-defective mutant (S171A/S275A) of CRTC2 localized in the nucleus and was constitutively active, which dramatically promoted HBV transcription and replication similar to FSK-treated wild-type CRTC2. Knock down of PGC1α, whose expression was induced by CRTC2, greatly compromised the enhancing effect of CRTC2 on HBV transcription and replication.
Our results clearly indicate that non-phosphorylated CRTC2 strongly enhances HBV biosynthesis through inducing PGC1α expression. Further study of the mechanisms will elucidate the importance of metabolic signals on HBV transcription and replication, and offer insight into potential targets for developing anti-HBV agents.
PMCID: PMC3940274  PMID: 24529027
HBV; CRTC2; Forskolin; Phosphorylation-defective CRTC2 mutant; PGC1α
6.  A Fasting Inducible Switch Modulates Gluconeogenesis Via Activator-Coactivator Exchange 
Nature  2008;456(7219):269-273.
During early fasting, increases in skeletal muscle proteolysis liberate free amino acids for hepatic gluconeogenesis in response to pancreatic glucagon. Hepatic glucose output diminishes during the late protein-sparing phase of fasting, when ketone body production by the liver supplies compensatory fuel for glucose-dependent tissues 1–4. Glucagon stimulates the gluconeogenic program by triggering the dephosphorylation and nuclear translocation of the CREB regulated transcription coactivator 2 (CRTC2; also known as TORC2), while parallel decreases in insulin signaling augment gluconeogenic gene expression through the de-phosphorylation and nuclear shuttling of Forkhead Box O1 (FOXO1) 5–7. Here we show that a fasting-inducible switch, consisting of the histone acetyl-transferase (HAT) P300 and the nutrient-sensing deacetylase Sirtuin 1 (SIRT1), maintains energy balance through the sequential induction of CRTC2 and FOXO1. Following glucagon induction, CRTC2 stimulated gluconeogenic gene expression through an association with P300, which we show here is also activated by de-phosphorylation at Ser89 during fasting. In turn, P300 increased hepatic CRTC2 activity by acetylating it at Lys628, a site that also targets CRTC2 for degradation following its ubiquitination by the E3 ligase Constitutive Photomorphogenic Protein (COP1) 8. Glucagon effects were attenuated during late fasting, when CRTC2 was down-regulated due to SIRT1-mediated deacetylation and when FOXO1 supported expression of the gluconeogenic program. Disrupting SIRT1 activity, by liver-specific knockout of the SIRT1 gene or by administration of SIRT1 antagonist, increased CRTC2 activity and glucose output, while exposure to SIRT1 agonists reduced them. In view of the reciprocal activation of FOXO1 and its coactivator peroxisome proliferator activated receptor gamma coactivator 1 alpha (PGC-1α) by SIRT1 activators 9–12, our results illustrate how the exchange of two gluconeogenic regulators during fasting maintains energy balance.
PMCID: PMC2597669  PMID: 18849969
7.  The CRTC1-NEDD9 Signaling Axis Mediates Lung Cancer Progression Caused by LKB1 Loss 
Cancer research  2012;72(24):6502-6511.
Somatic mutation of the tumor suppressor gene LKB1 occurs frequently in lung cancer where it causes tumor progression and metastasis, but the underlying mechanisms remain mainly unknown. Here, we show that the oncogene NEDD9 is an important downstream mediator of lung cancer progression evoked by LKB1 loss. In de novo mouse models, RNAi-mediated silencing of Nedd9 inhibited lung tumor progression, whereas ectopic NEDD9 expression accelerated this process. Mechanistically, LKB1 negatively regulated NEDD9 transcription by promoting cytosolic translocation of CRTC1 from the nucleus. Notably, ectopic expression of either NEDD9 or CRTC1 partially reversed the inhibitory function of LKB1 on metastasis of lung cancer cells. In clinical specimens, elevated expression of NEDD9 was associated with malignant progression and metastasis. Collectively, our results decipher the mechanism through which LKB1 deficiency promotes lung cancer progression and metastasis, and provide a mechanistic rationale for therapeutic attack of these processes.
PMCID: PMC3755891  PMID: 23074285
8.  Glucagon regulates gluconeogenesis through KAT2B- and WDR5-mediated epigenetic effects 
The Journal of Clinical Investigation  2013;123(10):4318-4328.
Circulating pancreatic glucagon is increased during fasting and maintains glucose balance by stimulating hepatic gluconeogenesis. Glucagon triggering of the cAMP pathway upregulates the gluconeogenic program through the phosphorylation of cAMP response element–binding protein (CREB) and the dephosphorylation of the CREB coactivator CRTC2. Hormonal and nutrient signals are also thought to modulate gluconeogenic gene expression by promoting epigenetic changes that facilitate assembly of the transcriptional machinery. However, the nature of these modifications is unclear. Using mouse models and in vitro assays, we show that histone H3 acetylation at Lys 9 (H3K9Ac) was elevated over gluconeogenic genes and contributed to increased hepatic glucose production during fasting and in diabetes. Dephosphorylation of CRTC2 promoted increased H3K9Ac through recruitment of the lysine acetyltransferase 2B (KAT2B) and WD repeat–containing protein 5 (WDR5), a core subunit of histone methyltransferase (HMT) complexes. KAT2B and WDR5 stimulated the gluconeogenic program through a self-reinforcing cycle, whereby increases in H3K9Ac further potentiated CRTC2 occupancy at CREB binding sites. Depletion of KAT2B or WDR5 decreased gluconeogenic gene expression, consequently breaking the cycle. Administration of a small-molecule KAT2B antagonist lowered circulating blood glucose concentrations in insulin resistance, suggesting that this enzyme may be a useful target for diabetes treatment.
PMCID: PMC3784539  PMID: 24051374
9.  Cytomegalovirus-induced salivary gland pathology: resistance to kinase inhibitors of the upregulated host cell EGFR/ERK pathway is associated with CMV-dependent stromal overexpression of IL-6 and fibronectin 
Herpesviridae  2013;4:1.
Recently we identified a relationship between human cytomegalovirus (hCMV) and human salivary gland (SG) mucoepidermoid carcinoma (MEC) in over 90% of cases; tumorigenesis in these cases uniformly correlated with active hCMV protein expression and an upregulation of the EGFR → ERK pathway. Our previously characterized, novel mouse organ culture model of mouse CMV (mCMV)-induced tumorigenesis displays a number of histologic and molecular characteristics similar to human MEC.
Newborn mouse submandibular glands (SMGs) were incubated with 1 × 105 PFU/ml of lacZ-tagged mCMV RM427+ on day 0 for 24 hours and then cultured in virus-free media for a total of 6 or 12 days with or without EGFR/ERK inhibitors and/or aciclovir. SMGs were collected for histology, immunolocalization (pERK, FN, IL-6), viral distribution, or Western blot analysis (pERK).
Here we report: (1) mouse SMG tumors soon exhibit an acquired resistance to EGFR/ERK pathway kinase inhibitors, alone or in combination; (2) long term tumor regression can only be sustained by concurrent inhibitor and antiviral treatment; (3) CMV-dependent, kinase inhibitor resistance is associated with overexpression of fibronectin and IL-6 proteins in abnormal stromal cells.
Acquired resistance to kinase inhibitors is dependent upon CMV dysregulation of alternative pathways with downstream effectors common with the targeted pathway, a phenomenon with important therapeutic implications for human MEC of salivary glands.
PMCID: PMC3602079  PMID: 23342981
Cytomegalovirus; Salivary gland; Tumorigenesis; EGFR; ERK; Fibronectin; Interleukin 6
10.  The CREB Coactivator CRTC2 Links Hepatic ER Stress and Fasting Gluconeogenesis 
Nature  2009;460(7254):534-537.
In fasted mammals, circulating pancreatic glucagon stimulates hepatic gluconeogenesis in part through the CREB Regulated Transcription Coactivator 2 (CRTC2; also referred to as TORC2) 1,2. Hepatic glucose production is elevated in obesity, reflecting chronic increases in endoplasmic reticulum (ER) stress that promote insulin resistance 3. Whether ER stress also modulates the gluconeogenic program directly, however, is unclear. Here we show that CRTC2 functions as a dual sensor for ER stress and fasting signals in liver. Acute increases in ER stress triggered the dephosphorylation and nuclear entry of CRTC2, which in turn promoted the expression of ER quality control genes through an association with Activating Transcription Factor 6 alpha (ATF6α), an integral branch of the unfolded protein response 4–9. In addition to mediating CRTC2 recruitment to ER stress inducible promoters, ATF6α also reduced hepatic glucose output by disrupting the CREB:CRTC2 interaction and thereby inhibiting CRTC2 occupancy over gluconeogenic genes. Conversely, hepatic glucose output was upregulated when hepatic ATF6α protein amounts were reduced, either by RNAi-mediated knockdown or as a result of persistent stress in obesity. As ATF6α over-expression in livers of obese mice reversed CRTC2 effects on the gluconeogenic program and lowered hepatic glucose output, our results demonstrate how cross-talk between ER stress and fasting pathways at the level of a transcriptional coactivator contributes to glucose homeostasis.
PMCID: PMC2730924  PMID: 19543265
11.  The CRTC1-SIK1 Pathway Regulates Entrainment of the Circadian Clock 
Cell  2013;154(5):1100-1111.
Retinal photoreceptors entrain the circadian system to the solar day. This photic resetting involves cAMP response element binding protein (CREB)-mediated upregulation of Per genes within individual cells of the suprachiasmatic nuclei (SCN). Our detailed understanding of this pathway is poor, and it remains unclear why entrainment to a new time zone takes several days. By analyzing the light-regulated transcriptome of the SCN, we have identified a key role for salt inducible kinase 1 (SIK1) and CREB-regulated transcription coactivator 1 (CRTC1) in clock re-setting. An entrainment stimulus causes CRTC1 to coactivate CREB, inducing the expression of Per1 and Sik1. SIK1 then inhibits further shifts of the clock by phosphorylation and deactivation of CRTC1. Knockdown of Sik1 within the SCN results in increased behavioral phase shifts and rapid re-entrainment following experimental jet lag. Thus SIK1 provides negative feedback, acting to suppress the effects of light on the clock. This pathway provides a potential target for the regulation of circadian rhythms.
Graphical Abstract
•Nocturnal light induces widespread transcriptional changes in the SCN•The CRTC1-SIK1 cascade regulates entrainment of the circadian clock•Negative feedback by SIK1 limits the effects of light on the clock•Homeostatic regulation of entrainment ensures gradual adaptation to a new time zone
A negative-feedback loop involving the kinase SIK1 and the transcriptional coactivator CRTC1 delays re-entrainment of the circadian clock to new time zones, causing jet lag. Remarkably, inhibition of SIK1 allows rapid re-entrainment after an experimental jet lag protocol in mice.
PMCID: PMC3898689  PMID: 23993098
12.  P63 Expression Can Be Used in Differential Diagnosis of Salivary Gland Acinic Cell and Mucoepidermoid Carcinomas 
Head and Neck Pathology  2012;7(1):64-68.
Differentiation of salivary gland acinic cell carcinoma from mucoepidermoid carcinoma can be diagnostically challenging as both may have prominent mucin production. P63 is a p53 homologue required for limb and epidermal morphogenesis. It is expressed in basal and myoepithelial cells of normal salivary gland tissues. In this immunohistochemical study, we examined the expression of p63 in salivary gland acinic cell and mucoepidermoid carcinomas (MEC) and its use in differentiating these two entities. A search was performed and appropriate cases were selected from Lifespan Hospital System archives as well as the consult archives of one author (DRG). 31 salivary gland acinic cell carcinomas (ACC) and 24 MEC were examined for p63 expression by immunohistochemistry. The nuclear immunoreactivity was examined by both authors and was graded semi-quantitatively with negative being less than 10 % of cells staining. Positive staining was graded as follows: 10–25 % of tumor cells staining was weakly positive, 26–75 % of tumor cells staining was moderately positive, and 76–100 % of tumor cells staining was strongly positive. Negative nuclear staining of the tumor cells was seen in 30/31 (96 %) of salivary gland ACC while 1/31 (3 %) showed diffuse nuclear staining of the tumor cells. This latter case was later reclassified as mammary analogue secretory carcinoma following confirmatory molecular testing for the ETV6-NTRK3 fusion gene. Strong positive nuclear staining of the tumor cells was seen in 24 (100 %) of salivary gland MEC cases. P63 is an immunohistochemical stain that can potentially aid in differentiating unusual ACC with prominent mucin production from MEC of the salivary gland. According to this study, acinic cell carcinoma is always negative for p63 immunoreactivity while mucoepidermoid carcinoma is always positive.
PMCID: PMC3597156  PMID: 23054955
Acinic cell carcinoma; Mucoepidermoid carcinoma; Mammary analogue secretory carcinoma; p63; Salivary gland
13.  Mdm2 controls CREB-dependent transactivation and initiation of adipocyte differentiation 
Cell Death and Differentiation  2012;19(8):1381-1389.
The role of the E3 ubiquitin ligase murine double minute 2 (Mdm2) in regulating the stability of the p53 tumor suppressor is well documented. By contrast, relatively little is known about p53-independent activities of Mdm2 and the role of Mdm2 in cellular differentiation. Here we report a novel role for Mdm2 in the initiation of adipocyte differentiation that is independent of its ability to regulate p53. We show that Mdm2 is required for cAMP-mediated induction of CCAAT/enhancer-binding protein δ (C/EBPδ) expression by facilitating recruitment of the cAMP regulatory element-binding protein (CREB) coactivator, CREB-regulated transcription coactivator (Crtc2)/TORC2, to the c/ebpδ promoter. Our findings reveal an unexpected role for Mdm2 in the regulation of CREB-dependent transactivation during the initiation of adipogenesis. As Mdm2 is able to promote adipogenesis in the myoblast cell line C2C12, it is conceivable that Mdm2 acts as a switch in cell fate determination.
PMCID: PMC3392627  PMID: 22388350
Mdm2; adipogenesis; CREB; Crtc2
14.  Embryonic salivary gland dysmorphogenesis in Twisted gastrulation deficient mice 
Archives of oral biology  2005;51(5):433-438.
Mouse Twisted gastrulation gene (Twsg1) expression is found throughout embryonic development, including substantial levels in the first branchial arch that gives rise to the submandibular salivary gland (SMG). We addressed the proposition that normal Twsg1 expression is critical to normal SMG ontogenesis.
Utilizing C57BL/6 embryos that were Twsg1−/− homozygotes, as well as wild type and heterozygote littermates, we investigated SMG development from gestational day 13 to newborn.
Twsg1 protein is immunodetected in epithelia throughout SMG development. Twsg1−/− embryos display widely variable craniofacial phenotypes that range from normal to severe holoprosencephaly/agnathia with no mandibular arch or stomodeum. The SMG phenotypes are correlated with the external craniofacial phenotype, ranging from normal to agenesis/aplasia.
It is evident that normal Twsg1 expression is critical for normal mouse SMG ontogenesis. Twsg1 loss of function is ultimately epistatic to the epigenome under normal physiologic conditions, but not always so. The reduced penetrance and variable expressivity seen in the SMGs of Twsg1−/− embryos is a challenging enigma.
PMCID: PMC1440928  PMID: 16289463
Twisted gastrulation gene; Mouse; Salivary glands; Development; Embryonic; BMP, bone morphogenetic protein; BSA, bovine serum albumin; FGF8, fibroblast growth factor 8; Pitx1, paired-like homeodomain transcription factor 1; Shh, sonic hedgehog; SMG, submandibular salivary gland; Tsg, twisted gastrulation; Twsg1, twisted gastrulation gene
15.  InsP3R-Ca2+ signaling takes center stage in the hormonal regulation of hepatic gluconeogenesis 
Cell Research  2012;22(11):1530-1532.
During fasting, dephosphorylation-dependent activation of the CREB coactivator CRTC2 by glucagon is crucial for activation of the hepatic gluconeogenic program, but the molecular mechanism by which hormones regulate CRTC2 activation remains unclear. A recent report in Nature showed that PKA-dependent phosphorylation of the inositol-1,4,5-trisphosphate receptor (InsP3R) induces Ca2+ mobilization, leading to increase in the phosphatase activity of calcineurin and the subsequent dephosphorylation of CRTC2, thereby resulting in the induction of gluconeogenic gene expression. It also showed that insulin-dependent phosphorylation of InsP3R by Akt inhibits Ca2+ mobilization and CRTC2 dephosphorylation, resulting in the suppression of gluconeogenesis.
PMCID: PMC3494392  PMID: 22710799
16.  CRTC2 (TORC2) Contributes to the Transcriptional Response to Fasting in the Liver but is Not Required for the Maintenance of Glucose Homeostasis 
Cell metabolism  2009;10(1):55-62.
The liver contributes to glucose homeostasis by promoting either storage or production of glucose depending on the physiological state. The cAMP response element binding protein (CREB) is a principal regulator of genes involved in coordinating the hepatic response to fasting, but its mechanism of gene activation remains controversial. We derived CRTC2-(CREB-regulated transcription coactivator 2; previously TORC2) deficient mice to assess the contribution of this cofactor to hepatic glucose metabolism in vivo. CRTC2 mutant hepatocytes showed reduced glucose production in response to glucagon, which correlated with decreased CREB binding to several gluconeogenic genes. However, despite attenuated expression of CREB target genes including PEPCK, G6Pase, and PGC1α, no hypoglycemia was observed in mutant mice. Collectively, these results provide genetic evidence supporting a role for CRTC2 in the transcriptional response to fasting, but indicate only a limited contribution of this cofactor to the maintenance of glucose homeostasis.
PMCID: PMC2748661  PMID: 19583954
17.  Human Kallikrein 8 Expression in Salivary Gland Tumors 
Head and Neck Pathology  2008;2(3):169-174.
The human kallikrein 8 protein (KLK8) is expressed in many normal tissues including esophagus, skin, testis, tonsil, kidney, breast, and salivary gland, and is found in biological fluids including breast milk, amniotic fluid, seminal fluid and serum. It has also been shown to be a biomarker and prognostic factor for breast cancer. The aim of this study was to determine whether KLK8 is expressed in salivary gland tissues and salivary gland tumors (both benign and malignant), in order to compare normal with tumor tissues. Pleomorphic adenomas, adenoid cystic carcinomas, polymorphous low grade adenocarcinomas, acinic cell carcinomas, mucoepidermoid carcinomas, and adenocarcinomas NOS of both minor and major salivary glands were examined. The results of this study indicate that most salivary gland tumors show high levels of expression of KLK8.
PMCID: PMC2807567  PMID: 20614312
Kallikreins; Human kallikrein 8; Salivary gland tumors; Prognostic markers; Immunohistochemistry
18.  GSK3β and CREB3 Gene Expression Profiling in Benign and Malignant Salivary Gland Tumors 
Iranian Biomedical Journal  2012;16(3):140-144.
Background: Salivary gland tumors (SGT) are rare lesions with uncertain histopathology. One of the major signaling pathways that participate in the development of several tumors is protein kinase A. In this pathway, glycogen synthase kinase β (GSK3β) and cAMP responsive element binding protein (CREB3) are two genes which are supposed to be down regulated in most human tumors. The expression level of the genes was evaluated in SGT to scrutinize their possible under expression in these tumors. Methods: Forty eight fresh tissue samples were obtained from patients with benign and malignant SGT, including pleomorphic adenoma, warthin’s tumor, mucoepidermoid carcinoma (MEC), salivary duct carcinoma and carcinoma ex pleomorphic adenoma. Eight normal samples were used as controls. Quantitative real-time PCR was used to analyze the expression level of interest genes. Results: Data was analyzed by statistical methods. GSK3β was downregulate in all samples and all results were statistically significant (P<0.05). CREB3 did not show a significant decrease or increase in its mRNA expression, but the results were significant in MEC and salivary duct carcinoma. Conclusion: GSK3β down regulation has been reported in many human tumors. This gene stimulates CREB3, inducing cell proliferation and oncogenesis. Our findings showed GSK3β down regulation; however, CREB3 expression level was close to normal group. No association between CREB3 expression and inactivated GSK3β could be postulated in SGT.
PMCID: PMC3629933  PMID: 23023215
Gene expression profiling; Salivary Gland neoplasms; glycogen synthase kinase 3 beta (GSK3β); CREB3 protein
19.  Localization of AQP5 during development of the mouse submandibular salivary gland 
Journal of Molecular Histology  2011;42(1):71-81.
Aquaporin 5 (AQP5) is known to be central for salivary fluid secretion. A study of the temporal-spatial distribution of AQP5 during submandibular gland (SMG) development and in adult tissues might offer further clues to its unknown role during development. In the present work, SMGs from embryonic day (E) 14.5–18.5 and postnatal days (P) 0, 2, 5, 25, and 60 were immunostained for AQP5 and analyzed using light microscopy. Additional confocal and transmission electron microscopy were performed on P60 glands. Our results show that AQP5 expression first occurs in a scattered pattern in the late canalicular stage and becomes more prominent and organized in the terminal tubuli/pro-acinar cells towards birth. Additional apical membrane staining in the entire intralobular duct is found just prior to birth. During postnatal development, AQP5 is expressed in both the luminal and lateral membrane of pro-acinar/acinar cells. AQP5 is also detected in the basal membrane of acinar cells at P25 and P60. In the intercalated ducts at P60, the male glands show apical staining in the entire segment, while only the proximal region is positive in the female glands. These results demonstrate an evolving distribution of AQP5 during pre- and postnatal development in the mouse SMGs.
PMCID: PMC3063871  PMID: 21203896
Submandibular gland; AQP5; Development; Prenatal; Postnatal; Sexual dimorphism
20.  The alveolar epithelial differentiation of glandular inner lining cells in a mucoepidermoid carcinoma of the lung: a case report 
Diagnostic Pathology  2012;7:137.
Mucoepidermoid carcinoma is a common malignant epithelial tumor of salivary glands, but relatively rare in lung. The histological features of mucoepidermoid carcinoma of the lung are similar to its counterpart arising from the salivary glands. Here, we reported a special tumor that occurred in the medial segment of the right lower lobe in a 22-year-old man. This tumor exhibited typical features of mucoepidermoid carcinoma with 3 cell types: squamoid cells, mucin-secreting cells and cells of intermediate type. These 3 types of cells organized into cysts, nests, glands and solid patterns. Specially, the inner lining cells of some glandular structures were uniform cuboidal and hobnail-like, similar to the alveolar epithelial cells. Immunohistochemistry staining revealed that the inner lining cells of glandular structures were positive for thyroid transcription factor-1 and surfactant protein-B, used as markers of alveolar epithelial cells, and were negative for p63. These findings for the first time demonstrated a rare alveolar epithelial differentiation of glandular inner lining cells in a mucoepidermoid carcinoma of the lung.
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PMCID: PMC3522998  PMID: 23043986
Mucoepidermoid carcinoma; Lung neoplasm; Thyroid transcription factor-1; Surfactant protein-B
21.  Differential Expression of p16INK4A and Cyclin D1 in Benign and Malignant Salivary Gland Tumors: A Study of 44 Cases 
Head and Neck Pathology  2013;7(3):224-231.
Salivary gland tumors (SGT) are a heterogeneous group of lesions. There is conflicting data concerning the molecular events involving the tumour suppressor retinoblastoma protein (pRb) pathway in these tumors. Few studies examined the alterations in components of the Rb pathway by immunohistochemical (IHC) methods in benign and malignant SGTs. Furthermore, recent evidence implicates human papillomavirus (HPV) in mucoepidermoid carcinoma (MEC) carcinogenesis. The purpose of our study is to examine p16INK4A and cyclin D1 expression in a variety of benign and malignant salivary gland tumors, and to investigate p16INK4A expression as a surrogate marker for HPV infection in MEC. Our series includes 30 malignant tumors [14 MEC, 6 acinic cell carcinomas (ACC), 5 polymorphous low grade adenocarcinomas (PLGA), 5 (AdCC)] and 14 benign tumors (4 benign cysts, 5 Warthin tumors and 5 pleomorphic adenomas (PA). All cases were tested by IHC for p16INK4A and cyclin D1. Testing for HPV wide spectrum (HPV-WS) was performed by in situ hybridization in all MEC cases. Staining intensity was recorded semi quantitatively (on a scale from 0 to 4+). Fisher’s exact test and Pearson X2 test with a p < 0.05 were used. Cyclin D1 and p16INK4A are expressed similarly in malignant and benign tumors (p = 0.146 and p = 0.543, respectively). None of the MEC cases showed nuclear reactivity for HPV-WS. Statistical analysis showed positive correlation between cyclin D1 and p16INK4A expression. Our findings suggest that p16INK4A overexpression is likely secondary to cyclin D1 gene upregulation or amplification. Further molecular studies are warranted.
PMCID: PMC3738749  PMID: 23315417
Cyclin D1; p16INK4A; Rb pathway; Salivary glands
22.  Epithelial Stem/Progenitor Cells in the Embryonic Mouse Submandibular Gland 
Frontiers of Oral Biology  2010;14:90-106.
Salivary gland organogenesis involves the specification, maintenance, lineage commitment, and differentiation of epithelial stem/progenitor cells. Identifying how stem/progenitor cells are directed along a series of cell fate decisions to form a functional salivary gland will be necessary for future stem cell regenerative therapy. The identification of stem/progenitor cells within the salivary gland has focused on their role in postnatal glands and little is known about them in embryonic glands. Here, we have reviewed the information available for other developing organ systems and used it to determine whether similar cell populations exist in the mouse submandibular gland. Additionally, using growth factors that influence salivary gland epithelial morphogenesis during development, we have taken a simple experimental approach asking whether any of these growth factors influence early developmental lineages within the salivary epithelium on a transcriptional level. These preliminary findings show that salivary epithelial stem/progenitor populations exist within the gland, and that growth factors that are reported to control epithelial morphogenesis may also impact cell fate decisions. Further investigation of the signaling networks that influence stem/progenitor cell behavior will allow us to hypothesize how we might induce autologous stem cells to regenerate damaged salivary tissue in a therapeutic context.
PMCID: PMC3375329  PMID: 20428013
23.  Comparative Cytokeratin Expression in the Different Cell Types of Salivary Gland Mucoepidermoid Carcinoma 
Head and Neck Pathology  2008;2(4):257-264.
Mucoepidermoid carcinoma is the most common malignant salivary gland tumor, composed of several different cell types, with controversial histogenesis. The aim of this study was to assess the expression of cytokeratins in mucoepidermoid carcinoma, comparing to cytokeratin expression in normal salivary glands, in order to establish a possible correlation between tumor cells immunostaining and mucoepidermoid carcinoma histogenesis and differentiation. Eighty cases of salivary gland mucoepidermoid carcinoma were immunohistochemically examined with the use of antibodies against cytokeratins 6, 7, 8, 13, 14, 18, and 19. Cytokeratin expression varied according to the cellular type: squamous cells presented high expression of cytokeratins 6, 7, 8, 14, 18, and 19; intermediate and mucous cells of cytokeratin 7; clear and columnar cells of cytokeratins 6, 7, 8 and the latter also expressed cytokeratin 18. Cytokeratin 13 expression was low in all cell types. Cytokeratin immunoexpression in mucoepidermoid carcinoma was variable according to the cellular type; but regardless of the cellular type studied, cytokeratins 7 and 13 were, respectively, constantly high and low expressed. The immunoprofile of the normal salivary glands was variable according to the component but, in general, cytokeratin profile in mucoepidermoid carcinoma showed similarity to the immunoexpression on the excretory duct unit of normal salivary glands.
PMCID: PMC2807572  PMID: 20614291
Mucoepidermoid carcinoma; Mucous cell; Intermediate cell; Squamous cell; Clear cell; Columnar cell; Oncocyitic cell; Histogenesis; Differentiation; Cytokeratin; Salivary glands; Immunohistochemistry
24.  Viral Gene Transfer to Developing Mouse Salivary Glands 
Journal of Dental Research  2012;91(2):197-202.
Branching morphogenesis is essential for the formation of salivary glands, kidneys, lungs, and many other organs during development, but the mechanisms underlying this process are not adequately understood. Microarray and other gene expression methods have been powerful approaches for identifying candidate genes that potentially regulate branching morphogenesis. However, functional validation of the proposed roles for these genes has been severely hampered by the absence of efficient techniques to genetically manipulate cells within embryonic organs. Using ex vivo cultured embryonic mouse submandibular glands (SMGs) as models to study branching morphogenesis, we have identified new vectors for viral gene transfer with high efficiency and cell-type specificity to developing SMGs. We screened adenovirus, lentivirus, and 11 types of adeno-associated viruses (AAV) for their ability to transduce embryonic day 12 or 13 SMGs. We identified two AAV types, AAV2 and bovine AAV (BAAV), that are selective in targeting expression differentially to SMG epithelial and mesenchymal cell populations, respectively. Transduction of SMG epithelia with self-complementary (sc) AAV2 expressing fibroblast growth factor 7 (Fgf7) supported gland survival and enhanced SMG branching morphogenesis. Our findings represent, to our knowledge, the first successful selective gene targeting to epithelial vs. mesenchymal cells in an organ undergoing branching morphogenesis.
PMCID: PMC3261122  PMID: 22095070
AAV; embryonic; gene transfer; organogenesis; salivary glands; transduction
25.  Human α-defensin (DEFA) gene expression helps to characterise benign and malignant salivary gland tumours 
BMC Cancer  2012;12:465.
Because of the infrequence of salivary gland tumours and their complex histopathological diagnosis it is still difficult to exactly predict their clinical course by means of recurrence, malignant progression and metastasis. In order to define new proliferation associated genes, purpose of this study was to investigate the expression of human α-defensins (DEFA) 1/3 and 4 in different tumour entities of the salivary glands with respect to malignancy.
Tissue of salivary glands (n=10), pleomorphic adenomas (n=10), cystadenolymphomas (n=10), adenocarcinomas (n=10), adenoidcystic carcinomas (n=10), and mucoepidermoid carcinomas (n=10) was obtained during routine surgical procedures. RNA was extracted according to standard protocols. Transcript levels of DEFA 1/3 and 4 were analyzed by quantitative realtime PCR and compared with healthy salivary gland tissue. Additionally, the proteins encoded by DEFA 1/3 and DEFA 4 were visualized in paraffin-embedded tissue sections by immunohistochemical staining.
Human α-defensins are traceable in healthy as well as in pathological altered salivary gland tissue. In comparison with healthy tissue, the gene expression of DEFA 1/3 and 4 was significantly (p<0.05) increased in all tumours – except for a significant decrease of DEFA 4 gene expression in pleomorphic adenomas and a similar transcript level for DEFA 1/3 compared to healthy salivary glands.
A decreased gene expression of DEFA 1/3 and 4 might protect pleomorphic adenomas from malignant transformation into adenocarcinomas. A similar expression pattern of DEFA-1/3 and -4 in cystadenolymphomas and inflamed salivary glands underlines a potential importance of immunological reactions during the formation of Warthin’s tumour.
PMCID: PMC3518101  PMID: 23050799
DEFA 1/3; DEFA 4; Salivary gland; Tumour; Defensins

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