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Pompe disease is a lysosomal storage disorder (LSD) caused by mutations in the gene that encodes acid alpha-glucosidase (GAA). Recently, small molecule pharmacological chaperones have been shown to increase protein stability and cellular levels for mutant lysosomal enzymes and have emerged as a new therapeutic strategy for the treatment of LSDs. In this study, we characterized the pharmacological chaperone 1-deoxynojirimycin (DNJ) on 76 different mutant forms of GAA identified in Pompe disease. DNJ significantly increased enzyme activity and protein levels for 16 different GAA mutants in patient-derived fibroblasts and in transiently transfected COS-7 cells. Additionally, DNJ increased the processing of these GAA mutants to their mature lysosomal forms, suggesting facilitated trafficking through the secretory pathway. Immunofluorescence microscopy studies showed increased colocalization of GAA with the lysosomal marker LAMP2 after incubation with DNJ, confirming increased lysosomal trafficking. Lastly, a GAA structural model was constructed based on the related eukaryotic glucosidase maltase-glucoamylase. The mutated residues identified in responsive forms of GAA are located throughout most of the structural domains, with half of these residues located in two short regions within the catalytic domain. Taken together, these data support further evaluation of DNJ as a potential treatment for Pompe disease in patients that express responsive forms of GAA.

Mesenchymal stem cells (MSCs) are located in postnatal bone marrow, show plasticity, are linked to various bone marrow disorders, exhibit phagocytosis, exert Ag-presenting properties (APC), and are immune suppressive. Unlike professional APCs, MSCs respond bimodally to IFN-γ in MHC-II expression, with expression at 10 U/ml and baseline, and down-regulation at 100 U/ml. The effects at high IFN-γ could not be explained by down-regulation of its receptor, IFN-γRI. In this study, we report on the mechanisms by which IFN-γ regulates MHC-II expression in MSCs. Gel shift assay and Western blot analyses showed dose-dependent increases in activated STAT-1, indicating responsiveness by IFN-γRI. Western blots showed decreased intracellular MHC-II, which could not be explained by decreased transcription of the master regulator CIITA, based on RT-PCR and in situ immunofluorescence. Reporter gene assays with PIII and PIV CIITA promoters indicate constitutive expression of PIII in MSCs and a switch to PIV by IFN-γ, indicating the presence of factors for effect promoter responses. We explained decreased MHC-II at the level of transcription because CIITA protein was observed in the cytosol and not in nuclei at high IFN-γ level. The proline/serine/threonine region of CIITA showed significant decrease in phosphorylation at high IFN-γ levels. An understanding of the bimodal effects could provide insights on bone marrow homeostasis, which could be extrapolated to MSC dysfunction in hematological disorders.

A significant concern in tissue repair is allo-rejection in a host with MHC-II mismatch. Since MSC appear to have veto properties to offset its own rejection, these stem cells may serve as ideal candidates in repair medicine. Interferon-g (IFN-g), which exacerbates immune responses, is known to enhance the expression of MHC-II in antigen presenting cells (e.g., macrophages). This led to the question on the mechanism by which MSC act as immune suppressor cells. We hypothesize that endogenous IFN-γ maintains basal MHC-II expression, while increased levels lead to its down regulation. Indeed, we observed a biphasic response of IFN-γ on the expression of MHC-II. While unstimulated MSC produced 19±5 pg/ml IFN-γ, exogenous IFN-γ (>100 U/ml) led to decreased expression of MHC-II. Suppression of IFN-g by siRNA resulted in undetectable MHC-II. The response of exogenous IFN-γ could not be explained by altered expression of IFN-g receptor type I (IFN-γRI) or changes in STAT-1 activation. However, western blots showed multiple isotypes of the MHC-II master regulator transcription factor, CIITA, in IFN-γ-stimulated MSC, with predominance of CIITA exhibiting low efficiency binding to DNA. This observation might explain why MHC-II is undetectable in the presence of high levels of IFN-γ. These findings support a potential for MSC as an adult stem cell in tissue repair.

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