Expression Of NDRG2 In Salivary Gland And Preliminary Study On Its Function

NDRG2 (N-myc down-stream regulated gene 2), a novel gene which was discovered and reported firstly by our research group. Although NDRG2 is possibly related to cell proliferation and differentiation, the exactly biological function has not yet been completely elucidated. Our previous findings have showed some interesting phenomena and which have important implications for our future research: NDRG2 is detected highly expressed in human salivary gland tissue by RNA dot blot assay; and immunoreactivity could be seen mainly in duct cells of sublingual gland in mice by the immunohistochemistry; yeast two-hybrid approach was used to screen the proteins which can interact with NDRG2, and of these 12 final positive clones, 3 fragments encoded Na~+/K~+-ATPaseβ1; Bioinformatics analysis revealed that there exist potential binding sites of ER (estrogen receptor) in the regulatory region of NDRG2.The main function of salivary duct cells is to reabsorb Na~+ and Cl- from saliva, and involved in the forming of normal saliva (low osmotic pressure), while the reabsorption of Na~+ depend on the epithelial sodium channel (ENaC). It has been reported that NDRG2 can activate ENAC in Xenopus laevis Oocytes. Na~+/K~+-ATPaseβ1 is a subunit of Na~+/K~+-ATPase (the other name is Na~+/K~+-pump), which is the indispensable part of the pump function. Na~+/K~+-ATPase locate on the basolateral side of salivary duct cells can pump Na~+ outside by expending energy, and this can form the Na~+ concentration difference, then effect the absorption of Na~+ on luminal side by ENaC from saliva which is excreted from acinus to duct. Oral dryness usually occurs in post-menopause female, more than 50% post-menopause female have conscious oral dryness, and the electrolyte ingredients of oral dryness patients have great discrepancy compared with normal persons, osmotic pressure and electrolyte concentration increase obviously and the saliva become very viscous.The foregoing research suggest intensively that NDRG2 play a very important role in salivary tissue, especially closely related with the forming of saliva, so more investigation and verification will be done in the present research.【Objectives】(1) To clear the distribution and localization of NDRG2 in salivary gland;(2) to investigate the function of NDRG2 in salivary gland and the correlation with Na~+/K~+-ATPaseβ1; (3) to probe whether NDRG2 was regulated by estrogen in transcription level; (4) to further analyze the relationship between estrogen and saliva forming, and the possible roles of NDRG2 involved in it.【Methods】(1) Immunohistochemistry and Immunofluorescence were used to detect the distribution and localization of NDRG2 in salivary gland tissue, and RT-PCR was applied to analyze the expression of NDRG1, NDRG2, NDRG3 and NDRG4 in human normal submaxillary gland. (2) Set up the salivary duct cell monolayer ion transport model to detect the effects of NDRG2 on the ions and H2O transport; the physical interaction between NDRG2 and Na~+/K~+-ATPaseβ1 were further detected with mammalian two-hybrid and co-immunoprecipitation; immunofluorescence was used to observe to the possible co-localization between NDRG2 and Na~+/K~+-ATPaseβ1; analyze the effect of NDRG2 to the activity of Na~+/K~+-ATPase; Real time PCR and Western Blot were applied to detect the effects of NDRG2 on the expression and function of Na~+/K~+-ATPaseβ1. (3) Real time PCR and Western Blot were used to detect the effect of 17β-estradiol (E2) on NDRG2 expression; and we assessed that whether NDRG2 was transcription regulated by E2-NDRG2 transcription complex by the dual report luciferase assay, ChIP and EMSA. (4) Ovariectomized rats were used to mimic estrogen level of the post-menopause female, and the variations of saliva flow, saliva electrolyte ingredients and related molecules NDRG2, Na~+/K~+-ATPase and ENaC expression in submaxillary gland were observed after ovariectomy.【Results】1. NDRG2 protein is specific highly expressed in the duct cell cytoplasm of salivary glandNDRG2 immunoreactivity could be seen mainly in duct cell cytoplasm of human three pairs of main salivary glands (parotid, submaxillary gland and sublingual gland) and rat submaxillary gland, while a very weak stain in acinus cells. The result of RT-PCR showed NDRG2 mRNA expression is high in human salivary gland, while the expression of NDRG1 and NDRG3 are relatively low, and NDRG4 is hardly expressed.2. NDRG2 binds and stabilizes Na~+/K~+-ATPaseβ1 proteinIn salivary duct cell monolayer ion transport model, NDRG2 were over expressed or silenced in HSG cells by using adenovirus or siRNA and the ratios of basolateral Na~+ concentration to apical Na~+ concentration is directly proportional to the NDRG2 protein level. And Cl- has the similar change as Na~+, while K~+ and Ca2~+ haven't obviously changes with NDRG2 expression change, meanwhile, the medium volume of both sides was not detected pronounced difference. The NDRG2-Na~+/K~+-ATPaseβ1 direct physical interaction was confirmed by mammalian two-hybrid and co-immunoprecipitation. NDRG2 and Na~+/K~+-ATPaseβ1 could co-localized in the perinuclear region of the cytoplasm in human immortalization salivary duct cell line—HSG. And we found Na~+/K~+-ATPase activity is directly proportional to NDRG2 protein level in the same total protein in HSG cells; and NDRG2 could increase the Na~+/K~+-ATPaseβ1 stability, and extend Na~+/K~+-ATPaseβ1 protein half-life.3. NDRG2 is regulated by E2-ERβtranscription complex, and NDRG2 is the target gene of estrogenE2 up-regulated NDRG2 expression in both mRNA and protein levels by time and dose dependent manners. Indirect immunofluorescence showed that ERαand ERβboth distributed in duct cells of salivary gland tissue, but the protein of ERβwas expressed much more than ERα, moreover, ERβand NDRG2 were both highly expressed in duct cells; ERβcould increase the activity of NDRG2 regulatory region report gene in a dose dependent manner, while ERαcouldn't activate report gene, and ERβspecific agonist DPN could activate NDRG2 report gene, while ERαspecific agonist PPT hadn't the same effect. ERβligand binding domain deletion mutant or silencing the expression of ERβby siRNA could both reduce the activity of NDRG2 report gene, and point mutation in potential ERE (estrogen response elements) reduced report gene remarkable. ChIP and EMSA assays showed exogenous or endogenous ERβcould bind with predicted ERE in NDRG2 regulatory region.4. NDRG2 involves in the oral dryness of ovariectomized rats The serum estradiol decreased quickly in ovariectomized rats, and ovariectomized rats appeared sparse hair and osteoporosis related with low estradiol level and showed apparent oral dryness symptoms including saliva flow diminishing, water intake increasing and saliva electrolyte ingredients Na~+ and Cl- concentration increasing. The protein level of NDRG2, Na~+/K~+-ATPaseβ1 and ENaCβin submaxillary gland were obviously decreased in ovariectomized rats as compared with control and sham operation animals, although the mRNA of Na~+/K~+-ATPaseβ1 wasn't changed remarkably.【Conclusions】We identified NDRG2 is one target gene of estrogen and provided the preliminary evidence that there exists the E2-ER-NDRG2-Na~+/K~+-ATPaseβ1 pathway and which is probably associated with post-menopause women oral dryness.