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 a serine/threonine phosphatase that is inhibited by the immunosuppressive drugs cyclosporine and FK506. Although calcineurin has been extensively studied in immune cells, less is known about calcineurin in other systems. There are two primary isoforms of the catalytic subunit of calcineurin, and mice have been created that lack either the alpha isoform (calcineurin A (CnA)alpha(-/-)) or the beta isoform (CnAbeta(-/-)). In this study, we examined the epidermis of CnAalpha(-/-) mice at birth and 4 weeks of age. Histological analyses revealed an attenuation of cells in the stratum spinosum of CnAalpha(-/-) mice. There was no significant difference in proliferation in the epidermis of CnAalpha(-/-) sections, but TUNEL assay revealed increased cell death in the supra-basal layers. Interestingly, the calcineurin substrate nuclear factor of activated T cells (NFATc) was highly expressed in the nucleus of basal epidermal cells in wild-type (WT) mice but was cytoplasmic in CnAalpha(-/-) mice, consistent with a loss of calcineurin activity. Moreover, NFATc activity was decreased in the epidermis of null mice compared with that in WT littermates. Finally, immunohistochemical staining revealed supra-basal expression of keratin 14 and decreased expression of differentiation-associated keratin 10 and involucrin. These findings suggest that calcineurin Aalpha activity is required for the normal differentiation and survival of epidermal cells.

These data suggest that monitoring specifically the β isoform may be more informative than non-isoform-specific assay methods.

Calcineurin is a serine/threonine phosphatase that is inhibited by the immunosuppressive agents cyclosporine A and FK506. Mice lacking the alpha isoform of the catalytic subunit of calcineurin (CnAα) have been created. On gross inspection, the skin of CnAα KO mice is noted to have decreased elasticity and resilience. Histological analyses reveal changes in the basal keratinocyte layer and an attenuation of cells in the supra‐basal layers, most notably the stratum spinosum. The calcineurin substrate NFATc is highly expressed in the nucleus of basal epidermal cells in WT mice but is cytoplasmic in KO mice, consistent with a loss of calcineurin activity in this layer. PCNA staining identified proliferating cells in the basal layer as expected, but TUNEL assay revealed increased cell death in the supra‐basal layers. Finally, immunohistochemical staining of keratins revealed that there is an increase in cells expressing the basal cell marker keratin 14 in the supra‐basal layers and a marked decrease in expression of the differentiated cell maker keratin 10 in CnAα KO mice. These findings suggest that calcineurin is required for normal differentiation of basal keratinocytes and survival of squamous epidermal cells. Interestingly, this model bears similarities to the inherited disorder Darier's disease and may shed light into the cellular basis of hyperproliferative conditions such as psoriasis.

Calcineurin is a calcium‐dependent enzyme that functions in many cells including the immune system, neurons, musculature and kidney. The phenotype of mice lacking the αisoform of the catalytic subunit (CnAα‐/‐) is notable for failure to thrive and early lethality; a cause for which has yet to be identified. In this study we report the rescue of CnAα‐/‐ mice to adulthood by feeding of a modified diet. The success of this approach suggested a defect in salivary gland function. Accordingly, while the rate of salivary production was normal, there was a significant decline in salivary amylase and peroxidase activity and lower amounts of sialic acid, indicating a defect in exocrine vesicle formation. Consistent with this finding, salivary glands from null mice demonstrated a marked attenuation of secretory vesicles and granular content of serosal acinar cells in submandibular glands. Subcellular fractionation of wildtype salivary glands revealed endoplasmic reticulum (ER) and trans‐Golgi network expression and activity of the αisoform. Loss of CnAα leads to retention of secretory vesicles in these fractions. Moreover, calcineurin activity in fractions containing the αisoform can be upregulated by acetylcholine and blocked by xestospongin, demonstrating a requirement for the IP3R. In conclusion, we find a critical role for CnAα downstream of the IP3R that is required for post‐ ER trafficking of secretory vesicles.