Molecular mechanisms of saliva secretion and hyposecretion.

The exocrine salivary gland secretes saliva, a fundamental body component to maintain oral homeostasis. Saliva is composed of water, ions, and proteins such as amylase, mucins, and immunoglobulins that play essential roles in the digestion of food, lubrication, and prevention of dental caries and periodontitis. An increasing number of people experience saliva hyposecretion due to aging, medications, Sjögren's syndrome, and radiation therapy for head and neck cancer. However, current treatments are mostly limited to temporary symptomatic relief. This review explores the molecular mechanisms underlying saliva secretion and hyposecretion to provide insight into putative therapeutic targets for treatment. Proteins implicated in saliva secretion pathways, including Ca2+ -signaling proteins, aquaporins, soluble N-ethylmaleimide-sensitive factor attachment protein receptors, and tight junctions, are aberrantly expressed and localized in patients with saliva hyposecretion, such as Sjögren's syndrome. Analysis of studies on the mechanisms of saliva secretion and hyposecretion suggests that crosstalk between fluid and protein secretory pathways via Ca2+ /protein kinase C and cAMP/protein kinase A regulates saliva secretion. Impaired crosstalk between the two secretory pathways may contribute to saliva hyposecretion. Future research into the detailed regulatory mechanisms of saliva secretion and hyposecretion may provide information to define novel targets and generate therapeutic strategies for saliva hyposecretion.

ΔNp63 maintains the fidelity of the myoepithelial cell lineage and directs cell differentiation programs in the murine salivary gland.

Salivary glands consist of several epithelial cell types of distinct lineages and functional characteristics that are established by directed differentiation programs of resident stem and progenitor cells. We have shown that ΔNp63, a crucial transcriptional regulator of stem/progenitor cells, is enriched in both the basal and myoepithelial cell (MEC) populations and that ΔNp63 positive cells maintain all the descendent epithelial cell lineages of the adult mouse salivary glands (mSGs). Although this pivotal role of ΔNp63 in driving the broader epithelial cell fate and identity in the mSG has been demonstrated, how ΔNp63 functions specifically in the commitment and differentiation of the MEC population is less understood. Using multiple genetic mouse models that allow for cell tracing, we show that ΔNp63 is critical in maintaining and renewing MECs, in part through the transcriptional regulation of Acta2 gene expression, a defining marker of this cell population. We demonstrate that during adult mSG homeostasis, ΔNp63 enriched MECs function as bipotent progenitor cells that maintain not only the MEC population, but also the distinctly different ductal cell lineages. The fidelity of this process is dependent on ΔNp63 expression, since MEC-specific ablation of ΔNp63 results in altered MEC differentiation and affects cellular plasticity resulting in aberrant differentiation of the intercalated ducts and acinar cells. In contrast, we find that the contribution of MECs to ductal and acinar cell regeneration following severe injury is independent of ΔNp63. Our observations offer new insights into cellular mechanisms driving MEC fate choices and differentiation programs in the context of salivary gland homeostasis and in response to injury and regeneration. Long term, these findings have implications for better treatment of salivary gland dysfunction through stem cell-based approaches.

The consequences of differential origin licensing dynamics in distinct chromatin environments.

Eukaryotic chromosomes contain regions of varying accessibility, yet DNA replication factors must access all regions. The first replication step is loading MCM complexes to license replication origins during the G1 cell cycle phase. It is not yet known how mammalian MCM complexes are adequately distributed to both accessible euchromatin regions and less accessible heterochromatin regions. To address this question, we combined time-lapse live-cell imaging with immunofluorescence imaging of single human cells to quantify the relative rates of MCM loading in euchromatin and heterochromatin throughout G1. We report here that MCM loading in euchromatin is faster than that in heterochromatin in early G1, but surprisingly, heterochromatin loading accelerates relative to euchromatin loading in middle and late G1. This differential acceleration allows both chromatin types to begin S phase with similar concentrations of loaded MCM. The different loading dynamics require ORCA-dependent differences in origin recognition complex distribution. A consequence of heterochromatin licensing dynamics is that cells experiencing a truncated G1 phase from premature cyclin E expression enter S phase with underlicensed heterochromatin, and DNA damage accumulates preferentially in heterochromatin in the subsequent S/G2 phase. Thus, G1 length is critical for sufficient MCM loading, particularly in heterochromatin, to ensure complete genome duplication and to maintain genome stability.

In this study the authors have, for the first time, quantified DNA replication origin licensing dynamics and distribution in single cells at subnuclear resolution. The cell cycle and DNA replication fields have long appreciated that origin licensing is both absolutely essential for replication and that licensing is strictly confined to G1 phase. The biochemical process of origin licensing- which is the DNA loading of MCM complexes- is known in considerable detail. What has never been explored in any system, is the dynamics of origin licensing itself. Here the authors define the dynamics of human MCM loading at different times within G1 in both euchromatin and heterochromatin, and explore the consequences of those dynamics for genome stability.

Transcriptomic and Single-Cell Analysis Reveals Regulatory Networks and Cellular Heterogeneity in Mouse Primary Sjögren's Syndrome Salivary Glands.

Sjögren's Syndrome (SS) is a chronic autoimmune disease of unknown etiology which primarily affects the salivary and lacrimal glands resulting in the loss of secretory function. Treatment options for SS have been hampered due to the lack of a better understanding of the underlying gene regulatory circuitry and the interplay between the myriad pathological cellular states that contribute to salivary gland dysfunction. To better elucidate the molecular nature of SS, we have performed RNA-sequencing analysis of the submandibular glands (SMG) of a well-established primary Sjögren's Syndrome (pSS) mouse model. Our comprehensive examination of global gene expression and comparative analyses with additional SS mouse models and human datasets, have identified a number of important pathways and regulatory networks that are relevant in SS pathobiology. To complement these studies, we have performed single-cell RNA sequencing to examine and identify the molecular and cellular heterogeneity of the diseased cell populations of the mouse SMG. Interrogation of the single-cell transcriptomes has shed light on the diversity of immune cells that are dysregulated in SS and importantly, revealed an activated state of the salivary gland epithelial cells that contribute to the global immune mediated responses. Overall, our broad studies have not only revealed key pathways, mediators and new biomarkers, but have also uncovered the complex nature of the cellular populations in the SMG that are likely to drive the progression of SS. These newly discovered insights into the underlying molecular mechanisms and cellular states of SS will better inform targeted therapeutic discoveries.

p63 and Its Target Follistatin Maintain Salivary Gland Stem/Progenitor Cell Function through TGF-ß/Activin Signaling.

Multipotent ΔNp63-positive cells maintain all epithelial cell lineages of the embryonic and adult salivary gland (SG). However, the molecular mechanisms by which ΔNp63 regulates stem/progenitor (SP) cell populations in the SG remains elusive. To understand the role of ΔNp63 in directing cell fate choices in this gland, we have generated ΔNp63-deleted adult mice and primary salivary cell cultures to probe alterations in SP cell differentiation and function. In parallel, we have leveraged RNA-seq and ChIP-seq-based characterization of the ΔNp63-driven cistrome and scRNA-seq analysis to molecularly interrogate altered SG cellular identities and differentiation states dependent on ΔNp63. Our studies reveal that ablation of ΔNp63 results in a loss of the SP cell population and skewed differentiation that is mediated by Follistatin-dependent dysregulated TGF-ß/Activin signaling. These findings offer new revelations into the SP cell gene regulatory networks that are likely to be relevant for normal or diseased SG states.

Transcriptomic and Network Analysis of Minor Salivary Glands of Patients With Primary Sjögren's Syndrome.

Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease characterized primarily by immune-mediated destruction of exocrine tissues, such as those of the salivary and lacrimal glands, resulting in the loss of saliva and tear production, respectively. This disease predominantly affects middle-aged women, often in an insidious manner with the accumulation of subtle changes in glandular function occurring over many years. Patients commonly suffer from pSS symptoms for years before receiving a diagnosis. Currently, there is no effective cure for pSS and treatment options and targeted therapy approaches are limited due to a lack of our overall understanding of the disease etiology and its underlying pathology. To better elucidate the underlying molecular nature of this disease, we have performed RNA-sequencing to generate a comprehensive global gene expression profile of minor salivary glands from an ethnically diverse cohort of patients with pSS. Gene expression analysis has identified a number of pathways and networks that are relevant in pSS pathogenesis. Moreover, our detailed integrative analysis has revealed a primary Sjögren's syndrome molecular signature that may represent important players acting as potential drivers of this disease. Finally, we have established that the global transcriptomic changes in pSS are likely to be attributed not only to various immune cell types within the salivary gland but also epithelial cells which are likely playing a contributing role. Overall, our comprehensive studies provide a database-enriched framework and resource for the identification and examination of key pathways, mediators, and new biomarkers important in the pathogenesis of this disease with the long-term goals of facilitating earlier diagnosis of pSS and to mitigate or abrogate the progression of this debilitating disease.

Genetic and scRNA-seq Analysis Reveals Distinct Cell Populations that Contribute to Salivary Gland Development and Maintenance.

Stem and progenitor cells of the submandibular salivary gland (SMG) give rise to, maintain, and regenerate the multiple lineages of mature epithelial cells including those belonging to the ductal, acinar, basal and myoepithelial subtypes. Here we have exploited single cell RNA-sequencing and in vivo genetic lineage tracing technologies to generate a detailed map of the cell fate trajectories and branch points of the basal and myoepithelial cell populations of the mouse SMG during embryonic development and in adults. Our studies show that the transcription factor p63 and alpha-smooth muscle actin (SMA) serve as faithful markers of the basal and myoepithelial cell lineages, respectively and that both cell types are endowed with progenitor cell properties. However, p63+ basal and SMA+ myoepithelial cells exhibit distinct cell fates by virtue of maintaining different cellular lineages during morphogenesis and in adults. Collectively, our results reveal the dynamic and complex nature of the diverse SMG cell populations and highlight the distinct differentiation potential of the p63 and SMA expressing subtypes in the stem and progenitor cell hierarchy. Long term these findings have profound implications towards a better understanding of the molecular mechanisms that dictate lineage commitment and differentiation programs during development and adult gland maintenance.

Functional characterization and genomic studies of a novel murine submandibular gland epithelial cell line.

A better understanding of the normal and diseased biology of salivary glands (SG) has been hampered, in part, due to difficulties in cultivating and maintaining salivary epithelial cells. Towards this end, we have generated a mouse salivary gland epithelial cell (mSGc) culture system that is well-suited for the molecular characterization of SG cells and their differentiation program. We demonstrate that mSGc can be maintained for multiple passages without a loss of proliferation potential, readily form 3D-spheroids and importantly express a panel of well-established salivary gland epithelial cell markers. Moreover, mSGc 3D-spheroids also exhibit functional maturation as evident by robust agonist-induced intracellular calcium signaling. Finally, transcriptomic characterization of mSGc by RNA-seq and hierarchical clustering analysis with adult organ RNA-seq datasets reveal that mSGc retain most of the molecular attributes of adult mouse salivary gland. This well-characterized mouse salivary gland cell line will fill a critical void in the field by offering a valuable resource to examine various mechanistic aspects of mouse salivary gland biology.

Docosahexaenoic acid inhibits IL-6 expression via PPARγ-mediated expression of catalase in cerulein-stimulated pancreatic acinar cells.

Cerulein pancreatitis mirrors human acute pancreatitis. In pancreatic acinar cells exposed to cerulein, reactive oxygen species (ROS) mediate inflammatory signaling by Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 3, and cytokine induction. Docosahexaenoic acid (DHA) acts as an agonist of peroxisome proliferator activated receptor γ (PPARγ), which mediates the expression of some antioxidant enzymes. We hypothesized that DHA may induce PPARγ-target catalase expression and reduce ROS levels, leading to the inhibition of JAK2/STAT3 activation and IL-6 expression in cerulein-stimulated acinar cells. Pancreatic acinar AR42J cells were treated with DHA in the presence or absence of the PPARγ antagonist GW9662, or treated with the PPARγ agonist troglitazone, and then stimulated with cerulein. Expression of IL-6 and catalase, ROS levels, JAK2/STAT3 activation, and nuclear translocation of PPARγ were assessed. DHA suppressed the increase in ROS, JAK2/STAT3 activation, and IL-6 expression induced nuclear translocation of PPARγ and catalase expression in cerulein-stimulated AR42J cells. Troglitazone inhibited the cerulein-induced increase in ROS and IL-6 expression, but induced catalase expression similar to DHA in AR42J cells. GW9662 abolished the inhibitory effect of DHA on cerulein-induced increase in ROS and IL-6 expression in AR42J cells. DHA-induced expression of catalase was suppressed by GW9662 in cerulein-stimulated AR42J cells. Thus, DHA induces PPARγ activation and catalase expression, which inhibits ROS-mediated activation of JAK2/STAT3 and IL-6 expression in cerulein-stimulated pancreatic acinar cells.

Docosahexaenoic Acid Induces Oxidative DNA Damage and Apoptosis, and Enhances the Chemosensitivity of Cancer Cells.

The human diet contains low amounts of ω-3 polyunsaturated fatty acids (PUFAs) and high amounts of ω-6 PUFAs, which has been reported to contribute to the incidence of cancer. Epidemiological studies have shown that a high consumption of fish oil or ω-3 PUFAs reduced the risk of colon, pancreatic, and endometrial cancers. The ω-3 PUFA, docosahexaenoic acid (DHA), shows anticancer activity by inducing apoptosis of some human cancer cells without toxicity against normal cells. DHA induces oxidative stress and oxidative DNA adduct formation by depleting intracellular glutathione (GSH) and decreasing the mitochondrial function of cancer cells. Oxidative DNA damage and DNA strand breaks activate DNA damage responses to repair the damaged DNA. However, excessive DNA damage beyond the capacity of the DNA repair processes may initiate apoptotic signaling pathways and cell cycle arrest in cancer cells. DHA shows a variable inhibitory effect on cancer cell growth depending on the cells' molecular properties and degree of malignancy. It has been shown to affect DNA repair processes including DNA-dependent protein kinases and mismatch repair in cancer cells. Moreover, DHA enhanced the efficacy of anticancer drugs by increasing drug uptake and suppressing survival pathways in cancer cells. In this review, DHA-induced oxidative DNA damage, apoptotic signaling, and enhancement of chemosensitivity in cancer cells will be discussed based on recent studies.

Phosphorylation and inactivation of glycogen synthase kinase 3ß (GSK3ß) by dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A).

Glycogen synthase kinase 3ß (GSK3ß) participates in many cellular processes, and its dysregulation has been implicated in a wide range of diseases such as obesity, type 2 diabetes, cancer, and Alzheimer disease. Inactivation of GSK3ß by phosphorylation at specific residues is a primary mechanism by which this constitutively active kinase is controlled. However, the regulatory mechanism of GSK3ß is not fully understood. Dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A) has multiple biological functions that occur as the result of phosphorylation of diverse proteins that are involved in metabolism, synaptic function, and neurodegeneration. Here we show that GSK3ß directly interacts with and is phosphorylated by Dyrk1A. Dyrk1A-mediated phosphorylation at the Thr(356) residue inhibits GSK3ß activity. Dyrk1A transgenic (TG) mice are lean and resistant to diet-induced obesity because of reduced fat mass, which shows an inverse correlation with the effect of GSK3ß on obesity. This result suggests a potential in vivo association between GSK3ß and Dyrk1A regarding the mechanism underlying obesity. The level of Thr(P)(356)-GSK3ß was higher in the white adipose tissue of Dyrk1A TG mice compared with control mice. GSK3ß activity was differentially regulated by phosphorylation at different sites in adipose tissue depending on the type of diet the mice were fed. Furthermore, overexpression of Dyrk1A suppressed the expression of adipogenic proteins, including peroxisome proliferator-activated receptor γ, in 3T3-L1 cells and in young Dyrk1A TG mice fed a chow diet. Taken together, these results reveal a novel regulatory mechanism for GSK3ß activity and indicate that overexpression of Dyrk1A may contribute to the obesity-resistant phenotype through phosphorylation and inactivation of GSK3ß.

Dyrk1A-mediated phosphorylation of RCAN1 promotes the formation of insoluble RCAN1 aggregates.

The mechanisms underlying aggregate formation in age-related neurodegenerative diseases remain not well understood. Here we investigated whether dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A (Dyrk1A) is involved in the formation of regulator of calcineurin 1 (RCAN1) aggregates. We show that RCAN1 self-associates and forms multimers, and that this process is promoted by the Dyrk1A-mediated phosphorylation of RCAN1 at the Thr(192) residue. Transgenic mice that overexpress the Dyrk1A exhibited lower levels of phospho-Thr(192)-RCAN1 in 10-month-old-group compared to littermate controls, when analyzed with soluble hippocampus lysates. These results suggest that the phosphorylation of RCAN1 by Dyrk1A stimulates the formation of insoluble aggregates upon aging.

Structures and properties of poly(3-alkylthiophene) thin-films fabricated though vapor-phase polymerization.

Organic semiconducting polymer thin-films of 3-hexylthiophene, 3-octylthiophene, 3-decylthiophene, containing highly oriented crystal were fabricated by gas-phase polymerization using the CVD technique. These poly(3-alkylthiophene) films had a crystallinity up to 80%, and possessed a Hall mobility up to 10 cm2/Vs. The degree of crystalinity and the mobility values increased as the alkyl chain length increased. The crystal structure of the polymers was composed of stacked layers constructed by a side-by-side arrangement of alkyl chains and in-plane pi-pi stacking. These thin films are capable of being applied to organic electronics as the active materials used in thin-film transistors and organic photovoltaic cells.