Summary Dysregulation of O‐GlcNAc modification catalyzed by O‐GlcNAc transferase (OGT) and O‐GlcNAcase (OGA) contributes to the etiology of chronic diseases of aging, including cancer, cardiovascular disease, type 2 diabetes, and Alzheimer’s disease. Here we found that natural aging in wild‐type mice was marked by a decrease in OGA and OGT protein levels and an increase in O‐GlcNAcylation in various tissues. Genetic disruption of OGA resulted in constitutively elevated O‐GlcNAcylation in embryos and led to neonatal lethality with developmental delay. Importantly, we observed that serum‐stimulated cell cycle entry induced increased O‐GlcNAcylation and decreased its level after release from G2/M arrest, indicating that O‐GlcNAc cycling by OGT and OGA is required for precise cell cycle control. Constitutively, elevated O‐GlcNAcylation by OGA disruption impaired cell proliferation and resulted in mitotic defects with downregulation of mitotic regulators. OGA loss led to mitotic defects including cytokinesis failure and binucleation, increased lagging chromosomes, and micronuclei formation. These findings suggest an important role for O‐GlcNAc cycling by OGA in embryonic development and the regulation of the maintenance of genomic stability linked to the aging process.

Materials and MethodsNeuronal Cell Cultures and Cytotoxicity. Primary cortical or hippocampal neuronal cultures were prepared as previously described (7). To induce excitotoxicity, the cells were prewashed with Tris-buffered control salt solution (CSS; 120 mM NaCl͞5.4 mM KCl͞1.8 mMCaCl 2 ͞25 mM Tris⅐HCl, pH 7.4͞15 mM glucose) and treated with CSS containing 300 M NMDA for 5 min. Toxicity was assayed 20-24 h after NMDA exposure by microscopic examination with computer-assisted cell counting. Total and dead cells were determined by nuclei staining with 100 ng͞ml 4Ј,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) (10 M), respectively. After a 10-min incubation, the cells were examined under a fluorescence microscope (Zeiss) with excitation at 360 nm. Cell death was determined as the ratio of dead to total cells and quantified by counting 1,000 cells. For staining of dead cells by terminal deoxynucleotidyltransferasemediated dUTP nick end labeling (TUNEL) assay, cells were fixed in 4% paraformaldehyde͞PBS and then stained by using a TUNEL Assay Kit (Molecular Probes) following protocols provided by the manufacturer. The cell death inhibitory effect of various agents was examined essentially as described (7,8).Western blotting was performed essentially as described (9).Focal Cerebral Ischemia Model. C57BL͞6 mice weighing 17-25 g were used for transient focal cerebral ischemia. After a midline neck incision, the left common carotid artery was isolated from the vagus nerve and ligated. The external carotid artery also was ligated, and the internal carotid artery was isolated carefully from the surrounding tissue. An 8-0 nylon filament (Ethicon, Somerville, NJ) was inserted into the common carotid artery through a small incision made in the proximity of the carotid bifurcation and advanced to the proximal part of the anterior cerebral artery to compromise the middle cerebral artery (MCA) flow. The filament was fixed in position by ligature. In sham-operated animals, the above procedures were performed except for the insertion of an intraluminal filament. For histological examinations, mice were perfused transcardially with heparinized PBS followed by 4% paraformaldehyde͞PBS for tissue fixation. Brains were removed and postfixed in 4% paraformaldehyde͞PBS at 4°C overnight. Coronal frozen sections (20 m) were prepared on a cryostat and stored at Ϫ80°C until use. The frozen sections were thawed, washed three times in PBS, permeabilized with 0.1% Triton X-100͞PBS at room temperature for 5 min, and then blocked in 5% skim milk͞3% BSA/PBS for 60 min. Subsequently, they were incubated with primary antibodies (1:200) at 4°C overnight and with secondary antibodies at room temperature for 2 h, and immunoreactivity was visualized by the avidin-biotin complex (ABC) method.Cell Lines and Cell Death Assays. HeLa cells, a human cervical carcinoma-derived cell line, were maintained in DMEM with 10% FBS, 2 mM L-glutamine, and 100 units of penicillin͞ streptomycin at 37°C with a 5% CO 2 atmosphere in a humidified incubator. P...

Two PDZ domain-containing proteins, NHERF and E3KARP are necessary for cAMP-dependent inhibition of Na ؉ /H ؉ exchanger 3 (NHE3). In this study, we demonstrate a specific role of E3KARP, which is not duplicated by NHERF, in Ca 2؉ -dependent inhibition of NHE3 activity. NHE3 activity is inhibited by elevation of intracellular Ca 2؉ ([Ca 2؉ ] i ) in PS120 fibroblasts stably expressing E3KARP but not those expressing NHERF. In addition, this Ca 2؉ -dependent inhibition requires Ca 2؉ -dependent association between ␣-actinin-4 and E3KARP. NHE3 is indirectly connected to ␣-actinin-4 in a protein complex through Ca 2؉ -dependent interaction between ␣-actinin-4 and E3KARP, which occurs through the actin-binding domain plus spectrin repeat domain of ␣-actinin-4. Elevation of [Ca 2؉ ] i results in oligomerization and endocytosis of NHE3 as well as in inhibition of NHE3 activity. Overexpression of ␣-actinin-4 potentiates the inhibitory effect of ionomycin on NHE3 activity by accelerating the oligomerization and endocytosis of NHE3. In contrast, overexpression of the actin-binding domain plus spectrin repeat domain acts as a dominant-negative mutant and prevents the inhibitory effect of ionomycin on NHE3 activity as well as the oligomerization and internalization of NHE3. From these results, we propose that elevated Ca 2؉ inhibits NHE3 activity through oligomerization and endocytosis of NHE3, which occurs via formation of an NHE3-E3KARP-␣-actinin-4 complex. Naϩ /H ϩ exchanger 3 (NHE3) 1 mediates the majority of NaCl and NaHCO 3 absorption in the ileum and proximal tubule of kidney (1-3). Elevation of [Ca 2ϩ ] i induced by several physiological and pathobiologic agonists (carbachol, serotonin, Escherichia coli heat-stable toxin b, rotavirus enterotoxin NSP5) inhibits NaCl absorption and brush border Na ϩ /H ϩ exchange activity in the small intestine and colon (4 -7). However, the effect of elevation of [Ca 2ϩ ] i in cell culture models differs among cell lines. In human colon cancer Caco-2 epithelial cells (C2bbe) stably transfected with NHE3, elevation of [Ca 2ϩ ] i by treatment with thapsigargin inhibited NHE3 activity (8). In contrast, elevating [Ca 2ϩ ] i by treatment with ionomycin did not alter NHE3 activity in PS120 fibroblasts (9), although basal [Ca 2ϩ ] i is involved in the regulation of NHE3 activity in these cells in a calmodulin-or calmodulin kinase II-dependent manner (10). These results therefore suggest that a regulatory factor, which is specifically expressed in ileum and Caco-2 (C2bbe) epithelial cells but not in PS120 fibroblasts, might be required for the Ca 2ϩ -dependent inhibition of NHE3 activity. To understand the mechanism of this inhibition, the molecular identity of the regulatory factors involved in the Ca 2ϩ -dependent inhibition of NHE3 activity needed to be elucidated.NHERF and E3KARP, two tandem PSD-95/Dlg-1/ZO-1 (PDZ) domain-containing proteins, were originally identified as regulatory proteins for protein kinase A (PKA)-dependent regulation of NHE3 (11-13). Both NHERF and E3KARP i...

Electroneutral NaCl absorption mediated by Na؉ /H ؉ exchanger 3 (NHE3) is important in intestinal and renal functions related to water/Na ؉ homeostasis.cGMP inhibits NHE3 in intact epithelia. However, unexpectedly it failed to inhibit NHE3 stably transfected in PS120 cells, even upon co-expression of cGMP-dependent protein kinase type II (cGKII). Additional co-expression of NHERF2, the tandem PDZ domain adapter protein involved in cAMP inhibition of NHE3, restored cGMP as well as cAMP inhibition, whereas NHERF1 solely restored cAMP inhibition. In vitro conditions were identified in which NHERF2 but not NHERF1 bound cGKII. The NHERF2 PDZ2 C terminus, which binds NHE3, also bound cGKII. A non-myristoylated mutant of cGKII did not support cGMP inhibition of NHE3. Although cGKI also bound NHERF2 in vitro, it did not evoke inhibition of NHE3 unless a myristoylation site was added. These results show that NHERF2, acting as a novel protein kinase G-anchoring protein, is required for cGMP inhibition of NHE3 and that cGKII must be bound both to the plasma membrane by its myristoyl anchor and to NHERF2 to inhibit NHE3.The rapid elevation of intestinal cAMP and cGMP levels by activation of adenylate cyclase and guanylate cyclase, respectively, inhibits intestinal NaCl absorption, either moderately as part of normal digestive physiology or excessively in diarrheal diseases. Some details of the mechanisms of acute regulation of intestinal NaCl absorption by cAMP are understood. Hormones such as vasoactive intestinal peptide or secretin and enterotoxins such as cholera toxin activate adenylate cyclase and increase cellular cAMP content. According to the current model, based on studies in PS120 fibroblasts and the polarized OK 1 renal proximal tubule cell line, acute elevation of cAMP inhibits NHE3 by stimulating its endocytosis plus decreasing its exocytosis and, additionally, by decreasing the NHE3 turnover number (1-4). NHE3 and cAMP-dependent protein kinase type II (PKAII) are part of the same signaling complex that is scaffolded by either of two brush border (BB)-associated PDZ domain containing proteins, NHERF1 (also called NHERF or EBP50) or NHERF2 (also called E3KARP) (5, 6). NHERF1/ NHERF2 each contain two homologous PDZ domains (PDZ1 and PDZ2) and an ERM (ezrin-radixin-moesin) binding domain, which anchors NHERF and its binding partners to the actin cytoskeleton via NHERF binding to ezrin. Ezrin binds both NHERF1/NHERF2 and PKAII and acts as a low affinity cAMP kinase-anchoring protein (AKAP), positioning PKAII so it can phosphorylate NHE3, which is required for cAMP inhibition of NHE3 (1, 6,7).In some cases, cGMP regulates intracellular events by mechanisms analogous to those demonstrated for cAMP. However, the effects of cGMP in the small intestine are not fully elucidated. The intrinsic ileal peptide guanylin and the Escherichia coli heat-stable enterotoxin (STa) both bind to the same BB receptor, guanylate cyclase-C, and within minutes increase intracellular cGMP content (8). STa, guanylin, and cGMP all rapidly i...