Abstract
SUMMARYThe severe pediatric disorder mucolipidosis II (ML-II; also known as I-cell disease) is caused by defects in mannose 6-phosphate (Man-6-P) biosynthesis. Patients with ML-II exhibit multiple developmental defects, including skeletal, craniofacial and joint abnormalities. To date, the molecular mechanisms that underlie these clinical manifestations are poorly understood. Taking advantage of a zebrafish model of ML-II, we previously showed that the cartilage morphogenesis defects in this model are associated with altered chondrocyte differentiation and excessive deposition of type II collagen, indicating that aspects of development that rely on proper extracellular matrix homeostasis are sensitive to decreases in Man-6-P biosynthesis. To further investigate the molecular bases for the cartilage phenotypes, we analyzed the transcript abundance of several genes in chondrocyte-enriched cell populations isolated from wild-type and ML-II zebrafish embryos. Increased levels of cathepsin and matrix metalloproteinase (MMP) transcripts were noted in ML-II cell populations. This increase in transcript abundance corresponded with elevated and sustained activity of several cathepsins (K, L and S) and MMP-13 during early development. Unlike MMP-13, for which higher levels of protein were detected, the sustained activity of cathepsin K at later stages seemed to result from its abnormal processing and activation. Inhibition of cathepsin K activity by pharmacological or genetic means not only reduced the activity of this enzyme but led to a broad reduction in additional protease activity, significant correction of the cartilage morphogenesis phenotype and reduced type II collagen staining in ML-II embryos. Our findings suggest a central role for excessive cathepsin K activity in the developmental aspects of ML-II cartilage pathogenesis and highlight the utility of the zebrafish system to address the biochemical underpinnings of metabolic disease.
Highlights
The autosomal recessive lysosomal disease mucolipidosis II (MLII; known as I-cell disease) is caused by defects in the biosynthesis of mannose 6-phosphate (Man-6-P) residues (Kollmann et al, 2010)
Cells isolated from Tg(fli1a:EGFP) zebrafish embryos express high levels of transcripts encoding extracellular matrix (ECM) proteins and ECM remodeling enzymes Transcript abundance profiling in isolated populations of zebrafish cells has emerged as an effective way to identify changes in gene expression that are associated with the development of specific cell types and tissues
In an effort to further explore the molecular basis of the cartilage phenotypes in the mucolipidosis II (ML-II) model, GFP-positive (GFP+) and -negative (GFP–) cells were isolated by fluorescence activated cell sorting (FACS) from dissociated WT and ML-II Tg(fli1a:EGFP) embryos, and quantitative real-time PCR analysis was performed on a targeted set of transcripts
Summary
The autosomal recessive lysosomal disease mucolipidosis II (MLII; known as I-cell disease) is caused by defects in the biosynthesis of mannose 6-phosphate (Man-6-P) residues (Kollmann et al, 2010). Many of the abnormalities associated with ML-II are noted at birth, highlighting the rapidly progressive nature of the disease and its impact on prenatal development (Sprigz et al, 1978; Herman and McAlister, 1996).
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