SUMMARY
Sex hormones and insulin have been implicated in articular cartilage metabolism. To supplement previous findings on the regulation of matrix synthesis with 17β-estradiol and insulin and to find a possible model to study cartilage metabolism in vitro, we evaluated the expression of estrogen receptors α and α (ERα, ERβ), androgen receptor (AR) and insulin receptor (IR), in immortalized C-28/I2 and T/C-28a2 chondrocytes and in human primary articular cartilage cells. Chondrocytes were treated with increasing concentrations of 17β-estradiol, dihydrotestosterone or insulin and analyzed by means of RT-PCR and Western blotting. Both cell lines as well as human articular chondrocytes expressed ER αand β, AR and IR at mRNA and protein levels. In immortalized C-28/I2 chondrocytes, we showed that increasing concentrations of 17β-estradiol diminished the 95 kDa band of IR. Since 17β-estradiol suppresses insulin-induced proline incorporation and type II collagen synthesis, as we have previously demonstrated, our findings give the first clue that 17β-estradiol may have negative effects on cartilage anabolism triggered by insulin during hormonal imbalance. Compared to chondrocytes cultured without hormones, immunostaining for ERα/β, AR and IR was decreased in both cell lines after incubation of cells with the receptor-specific hormones. It can be assumed that C-28/I2 and T/C-28a2 chondrocytes interact with the respective hormones. Our findings provide a reproducible model for investigating sex hormone and insulin receptors, which are present in low concentrations in articular chondrocytes, in the tissue-specific context of cartilage metabolism.
The bi-functional enzyme UDP-N-acetyl-2-epimerase/N-acetylmannosamine kinase (GNE) is the key enzyme of the sialic acid biosynthesis. Sialic acids are negatively charged nine carbon amino sugars and are found on most glycoproteins and many glycolipids in terminal positions, where they are involved in a variety of biological important molecular interactions. Inactivation of the GNE by homologous recombination results in early embryonic lethality in mice. Here, we report that GNE-deficient embryonic stem cells express less differentiation markers compared to wild-type embryonic stem cells. As a result, GNE-deficient embryonic stem cells fail to form proper embryoid bodies (EB) within the first day of culture. However, when culturing these cells in the presence of sialic acids for three days, also GNE-deficient embryonic stem cells form normal EBs. In contrast, when culturing these cells in sialic acid reduced medium, GNE-deficient embryonic stem cells proliferate faster and form larger EBs without any change in the expression of markers of the germ layers.
UDP‐N‐acetylglucosamine 2‐epimerase/N‐acetylmannosamine kinase (GNE) is the key enzyme for the biosynthesis of sialic acids. Sialic acids are terminal monosaccharides of glycoconjugates and gangliosides, which have an essential influence on various cell interactions. The sialylation of proteins varies during development, aging, and pathogenesis of degenerative diseases such as Morbus Alzheimer, diabetes mellitus type II, or myopathies. Mutation of methionine 743 in the GNE leads to a 30% reduction of the enzyme activity and is responsible for an aggressive form of GNE myopathy. GNE myopathy or hereditary inclusion body myopathy (HIBM) is an age‐dependent muscular dystrophy. Here, we analyzed the impact of the exchange of methionine to threonine at position 743 which introduces an additional potential phosphorylation/O‐GlcNAcylation site. We found increased O‐GlcNAcylation of the M743T variant compared to the wild‐type GNE. In addition, removal of the O‐GlcNAc of the M743T variant resulted in an increased activity comparable to activity of the wild‐type GNE. Furthermore, the half‐life of the M743T variant is two times longer than for the wild‐type GNE protein. This study provides that the balance of phosphorylation and O‐GlcNAcylation is decisive involved in efficiency and regulation of GNE.
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