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1.  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
2.  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
3.  Requirement of CRTC1 coactivator for hepatitis B virus transcription 
Nucleic Acids Research  2014;42(20):12455-12468.
Transcription of hepatitis B virus (HBV) from the covalently closed circular DNA (cccDNA) template is essential for its replication. Suppressing the level and transcriptional activity of cccDNA might have anti-HBV effect. Although cellular transcription factors, such as CREB, which mediate HBV transcription, have been well described, transcriptional coactivators that facilitate this process are incompletely understood. In this study we showed that CREB-regulated transcriptional coactivator 1 (CRTC1) is required for HBV transcription and replication. The steady-state levels of CRTC1 protein were elevated in HBV-positive hepatoma cells and liver tissues. Ectopic expression of CRTC1 or its homolog CRTC2 or CRTC3 in hepatoma cells stimulated the activity of the preS2/S promoter of HBV, whereas overexpression of a dominant inactive form of CRTC1 inhibited HBV transcription. CRTC1 interacts with CREB and they are mutually required for the recruitment to the preS2/S promoter on cccDNA and for the activation of HBV transcription. Accumulation of pregenomic RNA (pgRNA) and cccDNA was observed when CRTC1 or its homologs were overexpressed, whereas the levels of pgRNA, cccDNA and secreted HBsAg were diminished when CRTC1 was compromised. In addition, HBV transactivator protein HBx stabilized CRTC1 and promoted its activity on HBV transcription. Our work reveals an essential role of CRTC1 coactivator in facilitating and supporting HBV transcription and replication.
PMCID: PMC4227773  PMID: 25300488
4.  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
5.  Fusion Oncogenes in Salivary Gland Tumors: Molecular and Clinical Consequences 
Head and Neck Pathology  2013;7(Suppl 1):12-19.
Salivary gland tumors constitute a heterogeneous group of uncommon diseases that pose significant diagnostic and therapeutic challenges. However, the recent discovery of a translocation-generated gene fusion network in salivary gland carcinomas as well in benign salivary gland tumors opens up new avenues for improved diagnosis, prognostication, and development of specific targeted therapies. The gene fusions encode novel fusion oncoproteins or ectopically expressed normal or truncated oncoproteins. The major targets of the translocations are transcriptional coactivators, tyrosine kinase receptors, and transcription factors involved in growth factor signaling and cell cycle regulation. Notably, several of these targets or pathways activated by these targets are druggable. Examples of clinically significant gene fusions in salivary gland cancers are the MYB–NFIB fusion specific for adenoid cystic carcinoma, the CRTC1–MAML2 fusion typical of low/intermediate-grade mucoepidermoid carcinoma, and the recently identified ETV6–NTRK3 fusion in mammary analogue secretory carcinoma. Similarly, gene fusions involving the PLAG1 and HMGA2 oncogenes are specific for benign pleomorphic adenomas. Continued studies of the molecular consequences of these fusion oncoproteins and their down-stream targets will ultimately lead to the identification of novel driver genes in salivary gland neoplasms and will also form the basis for the development of new therapeutic strategies for salivary gland cancers and, perhaps, other neoplasms.
PMCID: PMC3712096  PMID: 23821214
Fusion oncogenes; Salivary gland neoplasms; Adenoid cystic carcinoma; Mucoepidermoid carcinoma; Biomarker; Targeted therapy; MYB–NFIB; CRTC1–MAML2; ETV6–NTRK3
6.  Regeneration of acinar cells following ligation of rat submandibular gland retraces the embryonic-perinatal pathway of cytodifferentiation 
Rat submandibular gland can regenerate following ligation-induced atrophy, eventually recovering its normal morphology and function. Previous studies have suggested that the regeneration process implies both self-proliferation of existing acini and formation of new acinar cells. One hypothesis is that new acinar cells may differentiate from the ductal cells in a similar fashion to the process of cytodifferentiation occurring during submandibular glandular development. In this study atrophy was induced, under recovery anaesthesia, by applying a metal clip on the main duct of the submandibular gland without including the chorda lingual nerve. After 2 weeks the duct was deligated for 3, 5 or 7 days or 8 weeks and the glands collected. Tissue was prepared for immunohistochemstry, biochemical analysis and RNA extraction. The histology of the regenerated glands shows several normal-looking acini, which have regained their glycoprotein content (AB/PAS positive), data also confirmed by biochemical analysis (SDS-PAGE/PAS). Regenerating tissue was characterized by the presence of embryonic-like branched structures ending with AB/PAS positive acinar cells. The proteins SMG-B and PSP are normally expressed in acinar cell precursors during development but only by intercalated ductal cells in the adult stage. In the adult regenerating gland mRNA levels of both SMG-B and PSP were found to be up-regulated compared to ligated glands and SMG-B expression localized to acinar cells whilst the ductal cells were negative. This study of rat submandibular gland regeneration suggests new acinar cells have differentiated from ducts and express markers of acinar cell precursors in a similar manner to the cytodifferentiation process occurring during glandular development.
PMCID: PMC2841285  PMID: 20056310
Salivary gland; Regeneration; SMG-B; PSP; Self-proliferation; Cell precursors
7.  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
8.  Characterization of tumorigenic cell lines from the recurrence and lymph node metastasis of a human salivary mucoepidermoid carcinoma 
Oral oncology  2013;49(11):10.1016/j.oraloncology.2013.08.004.
The long-term outcome of patients with mucoepidermoid carcinoma is poor. Limited availability of cell lines and lack of xenograft models is considered a major barrier to improved mechanistic understanding of this disease and development of effective therapies.
To generate and characterize human mucoepidermoid carcinoma cell lines and xenograft models suitable for mechanistic and translational studies.
Five human mucoepidermoid carcinoma specimens were available for generation of cell lines. Cell line tumorigenic potential was assessed by transplantation and serial in vivo passaging in immunodeficient mice, and cell line authenticity verified by short tandem repeat (STR) profiling.
A unique pair of mucoepidermoid carcinoma cell lines was established from a local recurrence (UM-HMC-3A) and from the metastatic lymph node (UM-HMC-3B) of the same patient, 4 years after surgical removal of the primary tumor. These cell lines retained epithelial-like morphology through 100 passages in vitro, contain the Crtc1-Maml2 fusion oncogene (characteristic of mucoepidermoid carcinomas), and express the prototypic target of this fusion (NR4A2). Both cell lines generated xenograft tumors when transplanted into immunodeficient mice. Notably, the xenografts exhibited histological features and Periodic Acid Schiff (PAS) staining patterns that closely resembled those found in human tumors. STR profiling confirmed the origin and authenticity of these cell lines.
These data demonstrate the generation and characterization of a pair of tumorigenic salivary mucoepidermoid carcinoma cell lines representative of recurrence and lymph node metastasis. Such models are useful for mechanistic and translational studies that might contribute to the discovery of new therapies for mucoepidermoid carcinoma.
PMCID: PMC3821871  PMID: 24035723
Mouse models; Salivary gland cancer; Xenograft; Oral cancer; Crtc1-Maml2; Tumor recurrence; Metastasis
9.  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
10.  Intracytoplasmic maturation of the human immunodeficiency virus type 1 reverse transcription complexes determines their capacity to integrate into chromatin 
Retrovirology  2006;3:4.
The early events of the HIV-1 life cycle include entry of the viral core into target cell, assembly of the reverse transcription complex (RTCs) performing reverse transcription, its transformation into integration-competent complexes called pre-integration complexes (PICs), trafficking of complexes into the nucleus, and finally integration of the viral DNA into chromatin. Molecular details and temporal organization of these processes remain among the least investigated and most controversial problems in the biology of HIV.
To quantitatively evaluate maturation and nuclear translocation of the HIV-1 RTCs, nucleoprotein complexes isolated from the nucleus (nRTC) and cytoplasm (cRTC) of HeLa cells infected with MLV Env-pseudotyped HIV-1 were analyzed by real-time PCR. While most complexes completed reverse transcription in the cytoplasm, some got into the nucleus before completing DNA synthesis. The HIV-specific RNA complexes could get into the nucleus when reverse transcription was blocked by reverse transcriptase inhibitor, although nuclear import of RNA complexes was less efficient than of DNA-containing RTCs. Analysis of the RTC nuclear import in synchronized cells infected in the G2/M phase of the cell cycle showed enrichment in the nuclei of RTCs containing incomplete HIV-1 DNA compared to non-synchronized cells, where RTCs with complete reverse transcripts prevailed. Immunoprecipitation assays identified viral proteins IN, Vpr, MA, and cellular Ini1 and PML associated with both cRTCs and nRTCs, whereas CA was detected only in cRTCs and RT was diminished in nRTCs. Cytoplasmic maturation of the complexes was associated with increased immunoreactivity with anti-Vpr and anti-IN antibodies, and decreased reactivity with antibodies to RT. Both cRTCs and nRTCs carried out endogenous reverse transcription reaction in vitro. In contrast to cRTCs, in vitro completion of reverse transcription in nRTCs did not increase their integration into chromatin.
These results suggest that RTC maturation occurs predominantly in the cytoplasm. Immature RTCs containing RT and incomplete DNA can translocate into the nucleus during mitosis and complete reverse transcription, but are defective for integration.
PMCID: PMC1360674  PMID: 16409631
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.  The Hippo signaling pathway is required for salivary gland development and its dysregulation is associated with Sjogren's-like disease 
Sjogren's syndrome (SS) is a complex autoimmune disease that primarily affects salivary and lacrimal glands and is associated with high morbidity. Although the prevailing dogma is that immune system pathology drives SS, increasing evidence points to structural defects, including defective E-cadherin adhesion, to be involved in its etiology. We have shown that E-cadherin plays pivotal roles in the development of the mouse salivary submandibular gland (SMG) by organizing apical-basal polarity in acinar and ductal progenitors and by signaling survival for differentiating duct cells. Recently, E-cadherin junctions have been shown to interact with effectors of the Hippo signaling pathway, a core pathway regulating organ size, cell proliferation and differentiation. We now show that Hippo signaling is required for SMG branching morphogenesis and is involved in the pathophysiology of SS. During SMG development, a Hippo pathway effector, TAZ, becomes increasingly phosphorylated and associated with E-cadherin and α-catenin, consistent with the activation of Hippo signaling. Inhibition of Lats2, an upstream kinase that promotes TAZ phosphorylation, results in dysmorphogenesis of the SMG and impaired duct formation. SMGs from NOD mice, a mouse model for SS, phenocopy the Lats2-inhibited SMGs and exhibit a reduction in E-cadherin junctional components, including TAZ. Importantly, labial specimens from human SS patients display mislocalization of TAZ from junctional regions to the nucleus, coincident with accumulation of extracellular matrix components, fibronectin and CTGF, known downstream targets of TAZ. Our studies show that Hippo signaling plays a crucial role in SMG branching morphogenesis and provide evidence that defects in this pathway are associated with SS in humans.
PMCID: PMC3864807  PMID: 24080911
E-cadherin adhesion; Hippo signaling; salivary gland; Sjogren's syndrome; TAZ
13.  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
14.  AID-Induced Genotoxic Stress Promotes B Cell Differentiation in the Germinal Center via ATM and LKB1 Signaling 
Molecular cell  2010;39(6):873-885.
During an immune response, B cells undergo rapid proliferation and AID-dependent remodeling of immunoglobulin (IG) genes within germinal centers (GCs) to generate memory B and plasma cells. Unfortunately, the genotoxic stress associated with the GC reaction also promotes most B cell malignancies. Here we report that exogenous- and intrinsic AID-induced DNA strand breaks activate ATM, which signals through an LKB1 intermediate to inactivate CRTC2, a transcriptional coactivator of CREB. Using genome-wide location analysis, we determined that CRTC2 inactivation unexpectedly represses a genetic program that controls GC B cell proliferation, self-renewal, and differentiation while opposing lymphomagenesis. Inhibition of this pathway results in increased GC B cell proliferation, reduced antibody secretion, and impaired terminal differentiation. Multiple distinct pathway disruptions were also identified in human GC B cell lymphoma patient samples. Combined, our data show that CRTC2 inactivation, via physiologic DNA damage response signaling, promotes B cell differentiation in response to genotoxic stress.
PMCID: PMC2945612  PMID: 20864035
15.  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
16.  Hunger and memory; CRTC coordinates long-term memory with the physiological state, hunger 
Animals form and store memory, which advantageously adjusts their behavior later on. Although the growing body of evidences suggests the basic mechanisms of memory, it is not clear whether and in which physiological state memory functions can be altered. Here we discuss our recent study that mild fasting facilitates long-term memory (LTM) formation in Drosophila.1 Canonical LTM in flies is induced by multiple training with rest intervals, and is mediated by a transcription factor, CREB and its binding protein, CBP. However, fasting allows LTM formation (fLTM) only by single-cycle training, in a manner dependent on another CREB binding protein, CRTC. Although it has been controversial, we are convinced that gene expression in a specific neural structure, called mushroom body (MB), is required for LTMs. We also showed data suggesting that reduced insulin signaling during fasting activates CRTC, thereby inducing fLTM formation. These data provides the conceptual advance that flies adapt their mechanisms for LTM formation according to their internal condition, hunger state. Due to limited food resources in the wild, fLTM could be one of the major form of LTM in natural environment. Furthermore, our data also indicate a novel conception that improvement of memory deficit might be achieved by activation of CRTC.
PMCID: PMC3829949  PMID: 24265850
long-term memory; Drosophila; CREB; CRTC; CBP; insulin signaling
17.  The CREB Coactivator CRTC3 Links Catecholamine Signaling to Energy Balance 
Nature  2010;468(7326):933-939.
Under lean conditions, the adipose-derived hormone leptin maintains energy balance in part through CNS-mediated increases in sympathetic outflow that enhance fat burning 1,2. Triggering of beta adrenergic receptors in adipocytes stimulates energy expenditure via cAMP-dependent increases in lipolysis and fatty acid oxidation 3. Although the underlying mechanism is unclear, catecholamine signaling in fat cells is thought to be disrupted in obesity 4, where it may contribute to the ectopic accumulation of lipid in liver and to the development of insulin resistance 5,6. Here we show that the cAMP responsive CREB coactivator CRTC3 promotes obesity by attenuating beta adrenergic receptor signaling in adipose; mice with a knockout of the CRTC3 gene have increased energy expenditure, are resistant to diet induced obesity, and are protected from the development of hepatic steatosis under high fat diet feeding conditions. CRTC3 was activated in response to catecholamine signals, when it reduced adenyl cyclase activity by upregulating the expression of RGS2 7–9, a metabolic syndrome susceptibility gene 10, which we show here is also a direct target of CREB and CRTC3. RGS2 expression was down-regulated in adipocytes from CRTC3−/− mice, leading to increases in insulin and catecholamine signaling that enhanced glucose and fatty acid oxidation. As a common human CRTC3 variant (Ser72Asn), with increased transcriptional activity, is associated with several anthropometric indices of adiposity in two distinct Mexican-American cohorts, our results suggest that adipocyte CRTC3 may play a role in the development of obesity in this population.
PMCID: PMC3025711  PMID: 21164481
18.  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
19.  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
20.  Identification and validation of PROM1 and CRTC2 mutations in lung cancer patients 
Molecular Cancer  2014;13:19.
Genetic alterations could be responsible lung cancer, the leading cause of worldwide cancer death.
This study investigated gene mutations in a Han Chinese family of lung cancer using the whole genome exome sequencing and subsequent Sanger sequencing validation and then confirmed alteration of prominin 1(PROM1) and cyclic AMP-response element binding protein-regulated transcription co-activator2 (CRTC2) in blood samples of 343 sporadic lung cancer patients vs. 280 healthy controls as well as in 200 pairs of lung cancer and the corresponding normal tissues using PCR-restriction fragment length polymorphism and directed DNA sequencing of PCR products.
The data showed PROM1 (p. S281R) and CRTC2 (p. R379C) mutations, in 5 and 2 cases of these 343 sporadic lung cancer patients, respectively. Notably, these mutations were absent in the healthy controls. Furthermore, in the 200 lung cancer and the matched normal tissues, PROM1 mutation occurred in 3 patients (i.e., one squamous cell carcinoma and two adenocarcinomas) with a mutation frequency of 1.5%, while CRTC2 mutation occurred in 5 patients (two squamous cell carcinomas and three adenocarcinomas) with a mutation frequency of 2.5%.
The data from the current study demonstrated novel PROM1 and CRTC2 mutations, which could promote lung cancer development.
PMCID: PMC4062918  PMID: 24484648
Lung cancer; Gene mutation; PROM1; CRTC2; Whole genome exome sequencing
21.  Salivary Gland NK Cells Are Phenotypically and Functionally Unique 
PLoS Pathogens  2011;7(1):e1001254.
Natural killer (NK) cells and CD8+ T cells play vital roles in containing and eliminating systemic cytomegalovirus (CMV). However, CMV has a tropism for the salivary gland acinar epithelial cells and persists in this organ for several weeks after primary infection. Here we characterize a distinct NK cell population that resides in the salivary gland, uncommon to any described to date, expressing both mature and immature NK cell markers. Using RORγt reporter mice and nude mice, we also show that the salivary gland NK cells are not lymphoid tissue inducer NK-like cells and are not thymic derived. During the course of murine cytomegalovirus (MCMV) infection, we found that salivary gland NK cells detect the infection and acquire activation markers, but have limited capacity to produce IFN-γ and degranulate. Salivary gland NK cell effector functions are not regulated by iNKT or Treg cells, which are mostly absent in the salivary gland. Additionally, we demonstrate that peripheral NK cells are not recruited to this organ even after the systemic infection has been controlled. Altogether, these results indicate that viral persistence and latency in the salivary glands may be due in part to the presence of unfit NK cells and the lack of recruitment of peripheral NK cells.
Author Summary
Human cytomegalovirus (HCMV) is a herpesvirus that infects 50–95% of human populations. In immunocompetent individuals, a primary infection often goes unnoticed and when resolved by the adaptive immune response, HCMV enters into a latent phase. The natural mouse pathogen murine CMV (MCMV) is a well-characterized animal model of viral infection that results in a non-replicative, chronic infection of an immunocompetent animal. MCMV is cleared efficiently by cytotoxic lymphocytes in all organs of the infected host, except the submandibular gland (SMG) of the salivary glands where it persists for several months eventually becoming latent for the life of the host. The acute response to this virus is dependent in part on natural killer (NK) cell cytotoxicity, as animals deficient in NK cells rapidly succumb to infection. Here, we identify a distinct salivary gland resident NK cell population, which detects the infection but remains mostly hyporesponsive. Peripheral NK cells, which control infection in the spleen, are not recruited to the salivary gland. Altogether, these data imply that CMV latency in the SMG could result from inadequate NK cell responses and can potentially lead to immune intervention to reverse CMV latency.
PMCID: PMC3020929  PMID: 21249177
22.  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
23.  GATA3 Immunohistochemical Expression in Salivary Gland Neoplasms 
Head and Neck Pathology  2013;7(4):311-315.
GATA3 is a zinc finger transcription factor that regulates the normal development of many tissues and cell types. Recent studies have shown that immunohistochemical nuclear staining for GATA3 among tumors is highly restricted to carcinomas of breast and urothelial origin; however salivary gland tumors have not been tested. Given that breast and salivary gland tissues are very similar with respect to embryologic development and structure, we performed GATA3 staining on a spectrum of salivary gland neoplasms. GATA3 immunohistochemistry was performed on a diverse collection of 180 benign and malignant salivary gland neoplasms including 10 acinic cell carcinomas, 2 adenocarcinomas not otherwise specified, 41 adenoid cystic carcinomas, 2 epithelial-myoepithelial carcinomas, 1 low grade cribriform cystadenocarcinoma, 15 mammary analogue secretory carcinomas, 7 metastatic squamous cell carcinomas, 27 mucoepidermoid carcinomas, 2 oncocytic carcinomas, 5 oncocytomas, 34 pleomorphic adenomas, 4 polymorphous low grade adenocarcinomas, 25 salivary duct carcinomas, and 5 Warthin tumors. Staining for GATA3 was observed in 92/180 (51 %) of salivary gland tumors. GATA3 staining was observed in most of the tumor types, but diffuse immunolabeling was consistently seen in salivary duct carcinoma (25 of 25) and mammary analogue secretory carcinoma (15 of 15)—the two tumor types that most closely resemble breast neoplasia. Background benign salivary gland tissue was also usually weakly positive in both acini and ducts. GATA3 immunostaining is not restricted to tumors of breast and urothelial origin. Rather, it is expressed across many different types of salivary gland neoplasms. As a result, salivary gland origin should be considered in the differential diagnosis of a GATA3-positive carcinoma, particularly in the head and neck. Although GATA3 immunohistochemistry is not helpful in resolving the differential diagnosis between a primary salivary gland neoplasm and metastatic breast cancer, it may have some utility in subtyping salivary gland tumors, particularly salivary duct carcinoma and mammary analogue secretory carcinoma.
PMCID: PMC3824803  PMID: 23604756
GATA3; Immunohistochemistry; Salivary glands; Salivary duct carcinoma; Mammary analogue secretory carcinoma
24.  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
25.  Expression and Localization of α-amylase in the Submandibular and Sublingual Glands of Mice 
Acta Histochemica et Cytochemica  2014;47(3):95-102.
In the major salivary glands of mice, acinar cells in the parotid gland (PG) are known to be the main site for the production of the digestive enzyme α-amylase, whereas α-amylase production in the submandibular gland (SMG) and sublingual gland (SLG), as well as the cell types responsible for α-amylase production, has been less firmly established. To clarify this issue, we examined the expression and localization of both the mRNA and protein of α-amylase in the major salivary glands of male and female mice by quantitative and histochemical methods. α-amylase mRNA levels were higher in the order of PG, SMG, and SLG. No sexual difference was observed in α-amylase mRNA levels in the PG and SLG, whereas α-amylase mRNA levels in the female SMG were approximately 30% those in the male SMG. Using in situ hybridization and immunohistochemistry, signals for α-amylase mRNA and protein were found to be strongly positive in acinar cells of the PG, serous demilune cells of the SLG, and granular convoluted tubule (GCT) cells of the male SMG, weakly positive in seromucous acinar cells of the male and female SMG, and negative in mucous acinar cells of the SLG. These results clarified that α-amylase is produced mainly by GCT cells and partly by acinar cells in the SMG, whereas it is produced exclusively by serous demilune cells in the SLG of mice.
PMCID: PMC4164700  PMID: 25320406
α-amylase; salivary gland; acinus; duct; sexual dimorphism; mouse

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