A Putative O-Linked ß-N-Acetylglucosamine Transferase Is Essential for Hormogonium Development and Motility in the Filamentous Cyanobacterium Nostoc punctiforme.

Most species of filamentous cyanobacteria are capable of gliding motility, likely via a conserved type IV pilus-like system that may also secrete a motility-associated polysaccharide. In a subset of these organisms, motility is achieved only after the transient differentiation of hormogonia, which are specialized filaments that enter a nongrowth state dedicated to motility. Despite the fundamental importance of hormogonia to the life cycles of many filamentous cyanobacteria, the molecular regulation of hormogonium development is largely undefined. To systematically identify genes essential for hormogonium development and motility in the model heterocyst-forming filamentous cyanobacterium Nostoc punctiforme, a forward genetic screen was employed. The first gene identified using this screen, designated ogtA, encodes a putative O-linked ß-N-acetylglucosamine transferase (OGT). The deletion of ogtA abolished motility, while ectopic expression of ogtA induced hormogonium development even under hormogonium-repressing conditions. Transcription of ogtA is rapidly upregulated (1 h) following hormogonium induction, and an OgtA-GFPuv fusion protein localized to the cytoplasm. In developing hormogonia, accumulation of PilA but not HmpD is dependent on ogtA Reverse transcription-quantitative PCR (RT-qPCR) analysis indicated equivalent levels of pilA transcript in the wild-type and ΔogtA mutant strains, while a reporter construct consisting of the intergenic region in the 5' direction of pilA fused to gfp produced lower levels of fluorescence in the ΔogtA mutant strain than in the wild type. The production of hormogonium polysaccharide in the ΔogtA mutant strain is reduced compared to that in the wild type but comparable to that in a pilA deletion strain. Collectively, these results imply that O-GlcNAc protein modification regulates the accumulation of PilA via a posttranscriptional mechanism in developing hormogonia.IMPORTANCE Filamentous cyanobacteria are among the most developmentally complex prokaryotes. Species such as Nostoc punctiforme develop an array of cell types, including nitrogen-fixing heterocysts, spore-like akinetes, and motile hormogonia, that function in dispersal as well as the establishment of nitrogen-fixing symbioses with plants and fungi. These symbioses are major contributors to global nitrogen fixation. Despite the fundamental importance of hormogonia to the life cycle of filamentous cyanobacteria and the establishment of symbioses, the molecular regulation of hormogonium development is largely undefined. We employed a genetic screen to identify genes essential for hormogonium development and motility in Nostoc punctiforme The first gene identified using this screen encodes a eukaryotic-like O-linked ß-N-acetylglucosamine transferase that is required for accumulation of PilA in hormogonia.

TGF-ß Stimulates Endochondral Differentiation after Denervation.

Transforming growth factor beta (TGF-ß) is a multifunctional protein that induces gene expression of cartilage-specific molecules, but its exact role in the process of chondrogenesis is unclear. Because recent studies suggest that TGF-ß can facilitate chondrogenic precursor cells differentiating into chondrocytes, we sought to determine whether TGF-ß prevents denervation-induced reduction of endochondral bone formation in an experimental model. Mice were treated daily with recombinant human TGF-ß1 (rhTGF-ß1) for 3 weeks. We found that rhTGF-ß1 not only prevented denervation-induced reduction of gene expression of type II collagen, type X collagen, aggrecan, Indian hedgehog, and parathyroid hormone-related peptide, but also synergized endochondral differentiation. These results demonstrate that short-term systemic administration of TGF-ß substantially prevents denervation-induced reduction of endochondral bone formation via stimulating endochondral differentiation. Potential therapeutic applications will be pursued in further studies that address the molecular biological mechanism of TGF-ß on endochodral bone formation after denervation in animal models.

Transforming Growth Factor Beta is regulated by a Glucocorticoid-Dependent Mechanism in Denervation Mouse Bone.

Bone growth and remodeling is inhibited by denervation in adults and children, resulting in alterations of linear growth and bone mass and increased risk for osteoporosis and pathologic fractures. Transforming growth factor beta (TGF-ß) isoforms are a key group of growth factors that enhance bone formation. To explore the relation between denervation-induced reduction of bone formation and TGF-ß gene expression, we measured mRNA levels of TGF-ß in denervation mouse bone and found decreased mRNA levels of TGF-ß1, TGF-ß2 and TGF-ß3. These changes were accompanied by diminishing weight loss, bone mineral density (BMD), trabecular thickness, trabecular separation and trabecular number of femur and lumbar, serum osteocalcin, total calcium, intact parathyroid hormone, and increased serum C telopeptide. Recombinant human TGF-ß1 (rhTGF-ß1) prevented denervation-induced reduction of BMD further supporting our hypothesis that denervation-induced reduction of bone formation is a result of inhibition of TGF-ß gene expression. In addition, antiprogestins RU 38486 blunted the denervation-induced decrease in mRNA levels of TGF-ß group, while dexamethasone (DEX) decreased TGF-ß group mRNA levels in normal mice. Furthermore, the denervated-mice exhibited a threefold increase in plasma corticosterone. These results suggest that denervation-induced reduction of bone formation may be regulated by glucocorticoids via inhibition of TGF-ß gene expression at least in part.