Publications

Loss of function mutations in the autoimmune regulator (Aire) gene in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy patients and mutant mice lead to autoimmune manifestations that segregate as a monogenic trait, but with wide variation in the spectrum of organs targeted. To investigate the cause of this variability, the Aire knockout mutation was backcrossed to mice of diverse genetic backgrounds. The background loci strongly influenced the pattern of organs that were targeted (stomach, eye, pancreas, liver, ovary, thyroid, and salivary gland) and the severity of the targeting (particularly strong on the nonobese diabetic background, but very mild on the C57BL/6 background). Autoantibodies mimicked the disease pattern, with oligoclonal reactivity to a few antigens that varied between Aire-deficient strains. Congenic analysis and a whole genome scan showed that autoimmunity to each organ had a distinctive pattern of genetic control and identified several regions that controlled the pattern of targeting, including the major histocompatibility complex and regions of Chr1 and Chr3 previously identified in controlling type 1 diabetes.

The extracellular Ca(2+)-sensing receptor (CaR) plays an essential role in mineral homeostasis. Studies to generate CaR-knockout (CaR(-/-)) mice indicate that insertion of a neomycin cassette into exon 5 of the mouse CaR gene blocks the expression of full-length CaRs. This strategy, however, allows for the expression of alternatively spliced CaRs missing exon 5 [(Exon5(-))CaRs]. These experiments addressed whether growth plate chondrocytes (GPCs) from CaR(-/-) mice express (Exon5(-))CaRs and whether these receptors activate signaling. RT-PCR and immunocytochemistry confirmed the expression of (Exon5(-))CaR in growth plates from CaR(-/-) mice. In Chinese hamster ovary or human embryonic kidney-293 cells, recombinant human (Exon5(-))CaRs failed to activate phospholipase C likely due to their inability to reach the cell surface as assessed by intact-cell ELISA and immunocytochemistry. Human (Exon5(-))CaRs, however, trafficked normally to the cell surface when overexpressed in wild-type or CaR(-/-) GPCs. Immunocytochemistry of growth plate sections and cultured GPCs from CaR(-/-) mice showed easily detectable cell-membrane expression of endogenous CaRs (presumably (Exon5(-))CaRs), suggesting that trafficking of this receptor form to the membrane can occur in GPCs. In GPCs from CaR(-/-) mice, high extracellular [Ca(2+)] ([Ca(2+)](e)) increased inositol phosphate production with a potency comparable with that of wild-type GPCs. Raising [Ca(2+)](e) also promoted the differentiation of CaR(-/-) GPCs as indicated by changes in proteoglycan accumulation, mineral deposition, and matrix gene expression. Taken together, our data support the idea that expression of (Exon5(-))CaRs may compensate for the loss of full-length CaRs and be responsible for sensing changes in [Ca(2+)](e) in GPCs in CaR(-/-) mice.

Mucus hypersecretion is a prominent manifestation in patients with chronic inflammatory airway diseases and contributes to their morbidity and mortality by plugging airways and causing recurrent infections. Human neutrophil elastase (HNE) exists in high concentrations (1-20 microM) in airway secretions of these patients and induces overproduction of MUC5AC mucin, a major component of airway mucus. Previous studies showed that HNE induces MUC5AC mucin production involving reactive oxygen species (ROS) generation and TGF-alpha-dependent epidermal growth factor receptor (EGFR) activation in human airway epithelial cells. However, the molecular mechanisms involved in these responses are not defined. TNF-alpha-converting enzyme (TACE) cleaves pro-TGF-alpha into soluble TGF-alpha and can be activated by ROS. We hypothesize that HNE activates TACE via ROS generation, resulting in cleavage of pro-TGF-alpha, EGFR activation, and MUC5AC mucin expression in airway epithelial cells. Here we show that in human airway epithelial cells HNE increases TGF-alpha release, EGFR phosphorylation, and MUC5AC mucin expression, effects that were attenuated by TACE inhibitor TAPI-1 and by specific knockdown of TACE expression with small interfering RNA, implicating TACE in HNE-induced responses. These responses to HNE were also reduced by pretreatment with ROS scavengers, implicating ROS. Furthermore, we show that HNE causes protein kinase C (PKC) activation and translocation from cytosol to plasma membrane; blockade of this effect by PKC inhibitors reduced HNE-induced ROS generation and other responses, implicating PKC. We conclude that HNE induces MUC5AC mucin expression via a cascade involving PKC-ROS-TACE in human airway epithelial cells.