Rescue of Calcineurin Aα−/− Mice Reveals a Novel Role for the α Isoform in the Salivary Gland
2011; Elsevier BV; Volume: 178; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2010.12.054
ISSN1525-2191
AutoresRamesh N. Reddy, Juan A. Pena, Brian R. Roberts, Stephen R. Williams, S. Russ Price, Jennifer L. Gooch,
Tópico(s)Neuropeptides and Animal Physiology
ResumoCalcineurin is an important signal transduction mediator in T cells, neurons, the heart, and kidneys. Recent evidence points to unique actions of the two main isoforms of the catalytic subunit. Although the β isoform is required for T-cell development, α is important in the brain and kidney. In addition, mice lacking α but not β suffer from failure to thrive and early mortality. The purpose of this study was to identify the cause of postnatal death of calcineurin α null (CnAα−/−) mice and to determine the mechanism of α activity that contributes to the phenotype. CnAα−/− mice and wild-type littermate controls were fed a modified diet and then salivary gland function and histology were examined. In vitro studies were performed to identify the mechanism of α action. Data show that calcineurin is required for normal submandibular gland function and secretion of digestive enzymes. Loss of α does not impair nuclear factor of activated T-cell activity or expression but results in impaired protein trafficking downstream of the inositol trisphosphate receptor. These findings show a novel function of calcineurin in digestion and protein trafficking. Significantly, these data also provide a mechanism to rescue to adulthood a valuable animal model of calcineurin inhibitor-mediated neuronal and renal toxicities. Calcineurin is an important signal transduction mediator in T cells, neurons, the heart, and kidneys. Recent evidence points to unique actions of the two main isoforms of the catalytic subunit. Although the β isoform is required for T-cell development, α is important in the brain and kidney. In addition, mice lacking α but not β suffer from failure to thrive and early mortality. The purpose of this study was to identify the cause of postnatal death of calcineurin α null (CnAα−/−) mice and to determine the mechanism of α activity that contributes to the phenotype. CnAα−/− mice and wild-type littermate controls were fed a modified diet and then salivary gland function and histology were examined. In vitro studies were performed to identify the mechanism of α action. Data show that calcineurin is required for normal submandibular gland function and secretion of digestive enzymes. Loss of α does not impair nuclear factor of activated T-cell activity or expression but results in impaired protein trafficking downstream of the inositol trisphosphate receptor. These findings show a novel function of calcineurin in digestion and protein trafficking. Significantly, these data also provide a mechanism to rescue to adulthood a valuable animal model of calcineurin inhibitor-mediated neuronal and renal toxicities. Calcineurin is most familiar as the target of immunosuppression drugs cyclosporine and tacrolimus. However, calcineurin is also a key signal transduction molecule in a variety of cell types. Genetic knockout of the two main, closely related isoforms of the catalytic subunit has led to a number of new observations that have added to our knowledge of calcineurin action in the immune system,1Zhang B.W. Zimmer G. Chen J. Ladd D. Li E. Alt F.W. Wiederrecht G. Cryan J. O'Neill E.A. Seidman C.E. Abbas A.K. Seidman J.G. T cell responses in calcineurin A alpha-deficient mice.J Exp Med. 1996; 183: 413-420Crossref PubMed Scopus (140) Google Scholar, 2Bueno O.F. Brandt E.B. Rothenberg M.E. Molkentin J.D. Defective T cell development and function in calcineurin Abeta-deficient mice.Proc Natl Acad Sci U S A. 2002; 99: 9398-9403Crossref PubMed Scopus (158) Google Scholar brain,3Zhuo M. Zhang W. Son H. Mansuy I. Sobel R. Seidman J. Kandel E. A selective role of calcineurin Aalpha in synaptic depotentiation in hippocampus.Proc Natl Acad Sci U S A. 1999; 96: 4650-4655Crossref PubMed Scopus (149) Google Scholar muscle,4Parsons S.A. Wilkins B.J. Bueno O.F. Molkentin J.D. Altered skeletal muscle phenotypes in calcineurin Aalpha and Abeta gene-targeted mice.Mol Cell Biol. 2003; 23: 4331-4343Crossref PubMed Scopus (138) Google Scholar, 5Parsons S.A. Millay D.P. Wilkins B.J. Bueno O.F. Tsika G.L. Neilson J.R. Liberatore C.M. Yutzey K.E. Crabtree G.R. Tsika R.W. Molkentin J.D. Genetic loss of calcineurin blocks mechanical overload-induced skeletal muscle fiber type switching but not hypertrophy.J Biol Chem. 2004; 279: 26192-26200Crossref PubMed Scopus (153) Google Scholar heart,6Bueno O.F. Wilkins B.J. Tymitz K.M. Glascock B.J. Kimball T.F. Lorenz J.N. Molkentin J.D. Impaired cardiac hypertrophic response in calcineurin Abeta-deficient mice.Proc Natl Acad Sci U S A. 2002; 99: 4586-4591Crossref PubMed Scopus (218) Google Scholar and kidney.7Reddy R.N. Knotts T.L. Roberts B.R. Molkentin J.D. Price S.R. Gooch J.L. Calcineurin A-beta is required for hypertrophy but not matrix expansion in the diabetic kidney.J Cell Mol Med. 2009; https://doi.org/10.1111/j.1582-4934.2009.00910.xCrossref PubMed Scopus (15) Google Scholar, 8Gooch J.L. Guler R.L. Barnes J.L. Toro J.J. Loss of calcineurin Aalpha results in altered trafficking of AQP2 and in nephrogenic diabetes insipidus.J Cell Sci. 2006; 119: 2468-2476Crossref PubMed Scopus (41) Google Scholar, 9Gooch J.L. Roberts B.R. Cobbs S.L. Tumlin J.A. Loss of the alpha-isoform of calcineurin is sufficient to induce nephrotoxicity and altered expression of transforming growth factor-beta.Transplantation. 2007; 83: 439-447Crossref PubMed Scopus (42) Google Scholar Importantly, calcineurin α null (CnAα−/−) and calcineurin β null (CnAβ−/−) mice have significant phenotypic distinctions, suggesting that the isoforms have unique functions. For example, the majority of CnAα−/− mice die 3 to 4 weeks after birth10Gooch J.L. Toro J.J. Guler R.L. Barnes J.L. Calcineurin A-alpha but not A-beta is required for normal kidney development and function.Am J Pathol. 2004; 165: 1755-1765Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar whereas CnAβ−/− mice reach maturity and are fertile.2Bueno O.F. Brandt E.B. Rothenberg M.E. Molkentin J.D. Defective T cell development and function in calcineurin Abeta-deficient mice.Proc Natl Acad Sci U S A. 2002; 99: 9398-9403Crossref PubMed Scopus (158) Google Scholar CnAβ−/− mice, however, are immunocompromised and graft-tolerant6Bueno O.F. Wilkins B.J. Tymitz K.M. Glascock B.J. Kimball T.F. Lorenz J.N. Molkentin J.D. Impaired cardiac hypertrophic response in calcineurin Abeta-deficient mice.Proc Natl Acad Sci U S A. 2002; 99: 4586-4591Crossref PubMed Scopus (218) Google Scholar whereas Rag−/− mice with CnAα−/−-reconstituted immune systems can still be immunosuppressed with cyclosporine.1Zhang B.W. Zimmer G. Chen J. Ladd D. Li E. Alt F.W. Wiederrecht G. Cryan J. O'Neill E.A. Seidman C.E. Abbas A.K. Seidman J.G. T cell responses in calcineurin A alpha-deficient mice.J Exp Med. 1996; 183: 413-420Crossref PubMed Scopus (140) Google Scholar These findings highlight an important distinction between the action of the isoforms and suggest that although CnAβ is the predominant isoform in the immune system, CnAα may be important in nonimmune tissues. Data from our laboratory and other investigators have shown that CnAβ acts through nuclear factor of activated T cell (NFAT)c whereas CnAα does not.9Gooch J.L. Roberts B.R. Cobbs S.L. Tumlin J.A. Loss of the alpha-isoform of calcineurin is sufficient to induce nephrotoxicity and altered expression of transforming growth factor-beta.Transplantation. 2007; 83: 439-447Crossref PubMed Scopus (42) Google Scholar, 11Kilka S. Erdmann F. Migdoll A. Fischer G. Weiwad M. The proline-rich N-terminal sequence of calcineurin Abeta determines substrate binding.Biochemistry. 2009; 48: 1900-1910Crossref PubMed Scopus (27) Google Scholar As such, the mechanism of CnAα action still is unknown. Previously, we reported that loss of CnAα but not CnAβ results in mislocalization of the water channel aquaporin 2 in the kidney collecting duct.8Gooch J.L. Guler R.L. Barnes J.L. Toro J.J. Loss of calcineurin Aalpha results in altered trafficking of AQP2 and in nephrogenic diabetes insipidus.J Cell Sci. 2006; 119: 2468-2476Crossref PubMed Scopus (41) Google Scholar Cameron et al12Cameron A.M. Steiner J.P. Roskams A.J. Ali S.M. Ronnett G.V. Snyder S.H. Calcineurin associated with the inositol 1,4,5-trisphosphate receptor-FKBP12 complex modulates Ca2+ flux.Cell. 1995; 83: 463-472Abstract Full Text PDF PubMed Scopus (449) Google Scholar showed that calcineurin can be immunoprecipitated with the inositol-3 phosphate receptor (IP3R) and the ryanodine receptor, whereas Guo et al13Guo L. Nakamura K. Lynch J. Opas M. Olson E.N. Agellon L.B. Michalak M. Cardiac-specific expression of calcineurin reverses embryonic lethality in calreticulin-deficient mouse.J Biol Chem. 2002; 277: 50776-50779Crossref PubMed Scopus (89) Google Scholar reported that overexpression of constitutively active CnAα in the heart rescued embryonic lethality of calreticulin null mice. Together, these findings led us to develop the hypothesis that CnAα plays a novel role as a downstream target of calcium release from endoplasmic reticulum (ER) calcium channels. Consistent with this model, IP3R type II−/−/III−/− mice were reported to share features with CnAα null pups including failure to thrive (FTT) and early lethality.14Futatsugi A. Nakamura T. Yamada M.K. Ebisui E. Nakamura K. Uchida K. Kitaguchi T. Takahashi-Iwanaga H. Noda T. Aruga J. Mikoshiba K. IP3 receptor types 2 and 3 mediate exocrine secretion underlying energy metabolism.Science. 2005; 309: 2232-2234Crossref PubMed Scopus (252) Google Scholar Futatsugi et al14Futatsugi A. Nakamura T. Yamada M.K. Ebisui E. Nakamura K. Uchida K. Kitaguchi T. Takahashi-Iwanaga H. Noda T. Aruga J. Mikoshiba K. IP3 receptor types 2 and 3 mediate exocrine secretion underlying energy metabolism.Science. 2005; 309: 2232-2234Crossref PubMed Scopus (252) Google Scholar identified a defect in the salivary gland that led to nutritional deficiencies in the double null pups. The mice could be rescued to adulthood by feeding the pups “predigested” chow. We reasoned that if our model of CnAα action downstream of ER calcium release was correct, a similar strategy also might rescue CnAα−/− pups. In this study we report the rescue of CnAα−/− mice to adulthood. Our data show that calcineurin is required for normal salivary gland function and early digestion. Moreover, we report a unique role for the α isoform downstream of the IP3R. Loss of this action results in altered vesicle trafficking in vivo and in vitro. Interestingly, this action of calcineurin is independent from NFATc regulation and provides yet another example of a nonimmune function of calcineurin that may be relevant to toxicities associated with clinical inhibition of calcineurin. Antibodies were obtained as follows: calcineurin Aα, calcineurin Aβ, Rab5B, Rab8A, NFATc1, NFATc2, NFATc3, and NFATc4 were from Santa Cruz Biotechnology (Santa Cruz, CA); and calnexin and GM130 were from BD Transduction Laboratories (San Diego, CA). Cyclosporine, brefeldin A, acetylcholine, and xestospongin were purchased from Sigma-Aldrich (St. Louis, MO). Mice were generated by Johnathan Seidman (Howard Hughes Medical Institute, Harvard Medical School, Boston, MA) as previously described1Zhang B.W. Zimmer G. Chen J. Ladd D. Li E. Alt F.W. Wiederrecht G. Cryan J. O'Neill E.A. Seidman C.E. Abbas A.K. Seidman J.G. T cell responses in calcineurin A alpha-deficient mice.J Exp Med. 1996; 183: 413-420Crossref PubMed Scopus (140) Google Scholar and have been maintained in our laboratory. Mice were maintained and bred at the Atlanta Veterans Administration Medical Center and all procedures were performed in accordance with an approved Institutional Animal Care and Use Committee protocol. Genotypes of offspring from heterozygous breeding pairs were determined by polymerase chain reaction using the following primers: 5′-GGCAGGAGAGTAAATTCTTGC-3′, 5′-GTGGAATTCTCTGGAGACAAACCACC-3′, and neo-5′-TCTTGATTCCCACTTTGTGGTTCTA-3′. CnAα−/− mice were created on a mixed genetic background.1Zhang B.W. Zimmer G. Chen J. Ladd D. Li E. Alt F.W. Wiederrecht G. Cryan J. O'Neill E.A. Seidman C.E. Abbas A.K. Seidman J.G. T cell responses in calcineurin A alpha-deficient mice.J Exp Med. 1996; 183: 413-420Crossref PubMed Scopus (140) Google Scholar Therefore, all experiments were performed with littermate controls. Likewise, littermate controls also were fed the modified gel diet for all experiments involving rescued CnAα−/− mice. Transgenic NFATc-luciferase reporter mice were obtained from Jeffery Molkentin (Cincinnati Children's Hospital, Cincinnati, OH) as described previously6Bueno O.F. Wilkins B.J. Tymitz K.M. Glascock B.J. Kimball T.F. Lorenz J.N. Molkentin J.D. Impaired cardiac hypertrophic response in calcineurin Abeta-deficient mice.Proc Natl Acad Sci U S A. 2002; 99: 4586-4591Crossref PubMed Scopus (218) Google Scholar, 15Wilkins B.J. Dai Y.S. Bueno O.F. Parsons S.A. Xu J. Plank D.M. Jones F. Kimball T.R. Molkentin J.D. Calcineurin/NFAT coupling participates in pathological, but not physiological, cardiac hypertrophy.Circ Res. 2004; 94: 110-118Crossref PubMed Scopus (615) Google Scholar and were crossed with CnAα+/− mice in our laboratory. Dissected salivary glands were homogenized and NFATc-mediated luciferase activity was measured using a commercial kit (Promega, Madison, WI). Briefly, tissue sections were homogenized with 1 μL lysis buffer/mg tissue and particulate matter separated by centrifugation. Luciferase assay reagent (100 μL) was added to 20 μL of supernatant and luminescence was measured for 10 seconds using an OptoComp luminometer (MGM Instruments, Hamden, CT). Results were normalized by subtracting values obtained from identically processed samples from NFATc-luciferase–negative littermate mice. Double heterozygous mice were obtained from Dr. Ju Chen (University of California, San Diego, San Diego, CA). Double heterozygous mice were bred to generate double-null and wild-type littermates. Salivary glands then were obtained for examination of calcineurin activity. Salivary glands were harvested and then either fixed in 10% formalin for paraffin embedding or 4 parts paraformalydehyde:1 part glutaraldehyde for plastic embedding. Paraffin tissues were sectioned at 4 μm and then mounted on glass slides for H&E staining or immunohistochemistry. Plastic-embedded tissues were cut ultra thin, mounted on glass slides, and then stained with toluidine blue. Paraffin-embedded tissue sections (4-μm thick) were prepared by dewaxing followed by quenching of endogenous peroxidases by incubation in 0.3% H2O2 in methanol for 30 minutes. Antigens were unmasked by incubation in 10 mmol/L citrate buffer at 80°C for 10 minutes. Sections then were rehydrated in PBS–0.1% bovine serum albumin for 15 minutes before addition of appropriate blocking serum for 15 minutes. Sections were incubated with primary antibodies (dilutions, 1:100 to 250) overnight in a humidified chamber at 4°C. The following day, sections were washed 3 times in PBS–0.1% bovine serum albumin and then incubated with biotinylated secondary antibodies (dilutions, 1:100) for 1 hour at room temperature. Bound antibody was identified by immunoperoxidase staining following the manufacturer's instructions (Vector Laboratories, Burlingame, CA). In addition, sections were counterstained with hematoxylin. Finally, coverslips were mounted with Permount (Sigma-Aldrich) and sections were viewed by bright-field microscopy. Saliva was collected using capillary tubes after injection of pilocarpine (1 mg/kg body weight) administered intramuscularly under xylazine and ketamine anesthesia. Serum and salivary amylase and blood urea nitrogen levels were measured using Reflotron test strips in a Reflotron benchtop analyzer (Roche, Indianapolis, IN). Peroxidase and lysozyme activities were measured using commercially available kits from Molecular Probes (Eugene, OR). Sialic acid was measured using a sialic acid quantitation kit (Sigma-Aldrich). Electrolytes (sodium, potassium, chloride, and bicarbonate) were measured using a clinical analyzer (Emory University Hospital, Atlanta, GA). Osmolality was measured using a WESCOR osmometer (Logan, UT). Total protein was measured using a bicinchoninic acid reagent (Bio-Rad, Hercules, CA). Calcineurin phosphatase activity was determined as previously described.16Roberts B. Pohl J. Gooch J.L. A fluorimetric method for determination of calcineurin activity.Cell Calcium. 2008; 43: 515-519Crossref PubMed Scopus (12) Google Scholar Briefly, the calcineurin substrate peptide RII was synthesized with a phospho-serine at residue 15 and an amino-terminus TAMRA fluorescent tag. In a 96-well plate, the labeled substrate was mixed in equal parts with reaction buffer and sample and allowed to incubate at 30°C for 10 minutes. Each well then was transferred to a 96-well plate coated with titanium-oxide (Glygen, Baltimore, MD), followed by gentle shaking to allow binding of phosphorylated substrate. Finally, supernatants containing unbound peptide then were moved to a new 96-well plate and the amount of dephosphorylated peptide was determined by fluorimetry at 485 nm excitation and 528 nm emission. Calcineurin activity then was determined by extrapolating fluorescence of experimental samples from a standard curve of purified calcineurin (Sigma-Aldrich). Salivary gland tissues or cells were harvested, mechanically disrupted, and then suspended in buffer containing 0.25 mol/L sucrose, 0.02 mol/L HEPES, and 1 mmol/L EDTA. Tissues also were disrupted using a Dounce homogenizer and centrifuged at 1000 × g for 10 minutes to pellet debris. Supernatants were layered on a sucrose gradient (OptiPrep; Sigma-Aldrich) and then centrifuged at 100,000 × g for 60 minutes until subcellular layers separated. Seven or 8 fractions from low density to high density were collected and protein concentrations were determined using the Bradford Method (Sigma-Aldrich). Fractions were characterized by Western blotting for protein markers of the ER, Golgi, trans-Golgi network, and vesicles as indicated. Antibodies were obtained from Santa Cruz Biotechnology or Chemicon (Billerica, MA). ParC5 cells were obtained from David Quissell and maintained as previously reported17Quissell D.O. Barzen K.A. Redman R.S. Camden J.M. Turner J.T. Development and characterization of SV40 immortalized rat parotid acinar cell lines.In Vitro Cell Dev Biol Anim. 1998; 34: 58-67Crossref PubMed Scopus (55) Google Scholar and were transfected with empty vector or constitutively active calcineurin Aα18Cobbs S.L. Gooch J.L. NFATc is required for TGFbeta-mediated transcriptional regulation of fibronectin.Biochem Biophys Res Commun. 2007; 362: 288-294Crossref PubMed Scopus (29) Google Scholar by electroporation. A total of 10 μg DNA was electroporated in 10 × 106 cells using a Bio-Rad GenePulser electroporater according to the manufacturer's instructions. Cells were re-plated, allowed to recover, and then expanded for subcellular fractionation. All statistical analyses were completed using GraphPad Prism software (La Jolla, CA). A P value of less than 0.05 was considered statistically significant. Unless otherwise stated, all comparisons were a two-tailed Student's t-test. CnAα−/− pups were placed on a modified diet consisting of powdered standard rodent chow suspended in 1.5% gelatin beginning at approximately 14 days of age. Wild-type littermate mice were treated identically. Figure 1 shows that the modified diet produced an immediate reversal of FTT in the null pups. Once on the diet, CnAα−/− mice showed a period of rapid weight gain and then maintained a normal body weight through 8 weeks of age. Adult mice then were grossly indistinguishable from littermates; male mice were found to be fertile (data not shown). Therefore, the next goals were to determine the reason CnAα−/− mice suffer FTT and the mechanism of CnAα action. Rescue of weight gain and body mass with supplemental feeding suggested that, similar to IP3R II−/−/III−/− mice, CnAα−/− mice suffer from a nutritional deficiency. First, CnAα−/− mice were observed to have normal swallowing ability (as evidenced by ingestion of milk by newborn pups), adequate mobility to access food, and normal-appearing dentition and tooth formation (data not shown). Next, saliva was obtained from CnAα−/− mice after stimulation with the reversible cholinesterase inhibitor pilocarpine. There was no difference in the amount or rate of saliva produced by wild-type (WT) and null mice (Figure 2, A and B) and there was no difference between male and female mice (data not shown). Saliva then was analyzed. First, salivary osmolality was increased in null mice, however, electrolyte composition and protein content were normal. Salivary urea was increased along with blood urea nitrogen and serum osmolality, consistent with renal insufficiency as previously reported (Table 1).10Gooch J.L. Toro J.J. Guler R.L. Barnes J.L. Calcineurin A-alpha but not A-beta is required for normal kidney development and function.Am J Pathol. 2004; 165: 1755-1765Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar Next, amylase, peroxidase, and lysozyme activities were measured and found to be decreased (changes in lysozyme activity did not reach significance). Finally, activity of the main mucosal enzyme sialic acid was significantly lower in null mice (Figure 2, C–F).Table 1Characterization of Salivary CompositionWild-typeCnAα−/−Osmolality (mg/dL)194 ± 10227 ± 6⁎P < 0.05 compared with wild-type;Sodium level (mg/mL)73.7 ± 6.872.3 ± 4.1Potassium level (mEq/L)29.7 ± 0.829.8 ± 0.8Chloride level (mEq/L)95.3 ± 3.292.1 ± 3.0Bicarbonate level (mEq/L)25.3 ± 2.525.8 ± 1.5Total protein level (mg/mL)0.58 ± 0.060.42 ± 0.05Salivary BUN level (mg/dL)13.6 ± 0.826.3 ± 4.8⁎P < 0.05 compared with wild-type;Serum amylase level (U/L)6.8 ± 0.46.5 ± 0.2Serum osmolality (mg/dL)312 ± 1335 ± 3⁎P < 0.05 compared with wild-type;Serum BUN level (mg/dL)27.3 ± 1.177.7 ± 7.5†P < 0.001 compared with WT.BUN, blood urea nitrogen. P < 0.05 compared with wild-type;† P < 0.001 compared with WT. Open table in a new tab BUN, blood urea nitrogen. Examination of pancreas from CnAα−/− mice revealed a normal appearance including islet formation, number, and size, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining revealed no increase in cell death (data not shown). Serum amylase and insulin levels also were unchanged in null mice and fecal trypsin-like enzyme activity was normal (data not shown). Together, these data suggest that changes in the salivary gland including vesicle formation and enzyme secretion are the most likely explanation for the nutritional deficiency and failure to thrive of CnAα−/− mice. As a target of immunosuppressant drugs including cyclosporine, the finding that loss of calcineurin can alter normal salivary gland function may have important clinical implications. For example, cyclosporine has long been noted to cause anorexia in animal models and in human beings, although no mechanism has been described previously. Figure 3A shows that rats treated with cyclosporine for 2 weeks gained significantly less weight than vehicle-treated rats. We therefore examined salivary gland function in WT mice treated with cyclosporine or vehicle (10% ethanol) and found that, similar to loss of CnAα, cyclosporine did not alter pilocarpine-stimulated saliva production (Figure 3B). However, cyclosporine treatment significantly reduced amylase and lysozyme activities compared with vehicle treatment. In addition, sialic acid levels were significantly lower with cyclosporine treatment (Figure 3, C–F). Next, salivary glands were obtained from WT and CnAα−/− littermates, stained with H&E, and examined by light microscopy. Figure 4 shows that although sublingual and parotid glands lacked obvious histologic defects (Figure 4, A and D vs B and E), submandibular glands from CnAα−/− mice were altered significantly (Figure 4, C vs F). Plastic-embedded, toluidine blue–stained sections further illustrated a defect decrease in secretory vesicle (Figure 4, G and H). Quantitation of these changes revealed significant decreases in vesicle number, mucosal acini cell size, and protein content of serosal acini (Figure 4, I and J). NFAT transcription factors are well-characterized substrates of calcineurin. To determine whether loss of CnAα altered NFAT, expression of all four calcium-responsive isoforms (NFATc1–c4) was examined by immunohistochemistry. Figure 5 shows that NFATc1 and NFATc4 are the predominant isoforms expressed in the submandibular gland (Figure 5, A and G). Although mucosal acini size is altered, expression of the two isoforms is maintained in CnAα−/− tissues (Figure 5, B and H). Likewise, activity of the ubiquitously expressed NFATc-driven luciferase reporter construct4Parsons S.A. Wilkins B.J. Bueno O.F. Molkentin J.D. Altered skeletal muscle phenotypes in calcineurin Aalpha and Abeta gene-targeted mice.Mol Cell Biol. 2003; 23: 4331-4343Crossref PubMed Scopus (138) Google Scholar, 5Parsons S.A. Millay D.P. Wilkins B.J. Bueno O.F. Tsika G.L. Neilson J.R. Liberatore C.M. Yutzey K.E. Crabtree G.R. Tsika R.W. Molkentin J.D. Genetic loss of calcineurin blocks mechanical overload-induced skeletal muscle fiber type switching but not hypertrophy.J Biol Chem. 2004; 279: 26192-26200Crossref PubMed Scopus (153) Google Scholar was unchanged in null salivary gland tissue (Figure 5I). In contrast, treatment of WT mice with cyclosporine for 3 days resulted in a decrease in NFAT activity (Figure 5J). These results suggest that the primary role of CnAα in the salivary gland is NFATc-independent.Figure 5Expression of NFAT isoforms and activity is unchanged in CnAα−/− salivary glands. NFATc isoform expression in WT (left) and CnAα−/− (right) submandibular tissue. NFATc1 (A and B), NFATc2 (C and D), NFATc3 (E and F), and NFATc4 (G and H) was determined by immunohistochemistry using specific antibodies. Data shown are representative of at least 2 independent experiments using four to six mice per group. Original magnification, ×200. I: Luciferase production in salivary glands from three WT and three CnAα−/− mice expressing a transgenic NFATc-responsive luciferase promoter construct.4Parsons S.A. Wilkins B.J. Bueno O.F. Molkentin J.D. Altered skeletal muscle phenotypes in calcineurin Aalpha and Abeta gene-targeted mice.Mol Cell Biol. 2003; 23: 4331-4343Crossref PubMed Scopus (138) Google Scholar J: Luciferase production in salivary glands from three WT mice treated with vehicle alone (10% ethanol) or cyclosporine (20 mg/kg) daily for 3 days. *P < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine the mechanism of CnAα action, salivary glands were obtained from null and WT littermate mice for in vitro analyses. First, total calcineurin activity was examined in lysates from WT and CnAα−/− salivary glands. Activity was reduced by approximately one third in CnAα−/− samples, suggesting that CnAβ contributes the majority of total calcineurin activity in salivary glands (Figure 6A). Next, seven high-density to low-density fractions were collected from salivary glands pooled from three mice of each genotype. Expression and activity of calcineurin isoforms was examined. Figure 6B shows that the α isoform is expressed in the highest density fractions 1 to 3. In contrast, expression of the β isoform is highest in fractions 5 and 6. In CnAα−/− tissue, expression of CnAβ was shifted toward fraction 7 whereas CnAα was undetectable, as expected. Calcineurin activity also was examined in the same subcellular fractions; activity in fractions 1 and 2 was reduced significantly in CnAα−/− tissue compared with WT. Consistent with changes in β expression, calcineurin activity was increased in fraction 7 in null tissues compared with WT (Figure 6C). Finally, to determine the intracellular compartment where α is expressed, subcellular fractions were characterized using makers of the ER, Golgi apparatus, trans-Golgi network, and intracellular vesicles (Figure 6D). In both wild-type and null samples, factions 1 to 3 showed significant expression of calnexin supporting the ER as the site of α localization. Interestingly, in null samples, distribution of proteins identifying post-ER structures is altered compared with wild-type samples. Specifically, peak GM130 expression is in fraction 2 rather than fraction 4 and Rab8 is decreased in fractions 1 and 2. More striking, Rab5 expression is observed in fractions 1 to 3 in null tissues but absent in these fractions in the wild-type samples. To determine whether calcineurin activity is required for normal vesicle trafficking, cultured fibroblasts were treated with cyclosporine or vehicle alone for 4 hours and then Rab5 localization was determined in fractionated lysates. Figure 7A shows that inhibition of calcineurin is sufficient to prompt redistribution of Rab5 expression to higher-density vesicles that also express calnexin. This was examined further in Par5C salivary gland cells. Immunofluorescence for Rab5 shows that vesicle localization is altered with cyclosporine A treatment (Figure 7, B vs C). Moreover, the pattern of vesicle distribution is comparable with ER localization induced by brefeldin A treatment (Figure 7D) and distinct from Golgi localization induced by room temperature incubation (Figure 7E). Finally, Rab5 distribution was examined in CnAα−/− and CnAβ−/− fibroblasts. Figure 7F shows that Rab5 localization is decreased in the highest-density fractions and shifted toward fractions that also express calnexin. In contrast, Rab5 expression in CnAβ−/− fractions remains highest in fraction 8. Inhibition of remaining α activity with cyclosporine A produced a shift of Rab5 expressi
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