Drosophila heparan sulfate 3-0 sulfotransferase B Null Mutant Is Viable and Exhibits No Defects in Notch Signaling

Heparan sulfate proteoglycans （HSPGs） are critically involved in a variety of biological events. The functions of HSPGs are determined by the nature of the core proteins and modifications of heparan sulfate （HS） glycosaminoglycan （GAG） chains. The distinct O-sulfo- transferases are important for nonrandom modifications at specific positions. Two HS 3-0 sulfotransferase （Hs3st） genes, Hs3st-A and Hs3st-B, were identified in Drosophila. Previous experiments using RNA interference （RNAi） suggested that Hs3st-B was required for Notch signaling. Here, we generated a null mutant of Hs3st-B via ends-out gene targeting and examined its role（s） in development. We found that homozygous Hs3st-B mutants have no neurogenic defects or alterations in the expression of Notch signaling target gene. Thus, our results strongly argue against an essential role for Hs3st-B in Notch signaling. Moreover, we have generated two independent Hs3st-A RNAi lines which worked to deplete Hs3st-A. Importantly, Hs3st-A RNAi combined with Hs3st-B mutant flies did not alter the expression of Notch signaling components, arguing that both Hs3st-A and Hs3st-B were not essential for Notch signaling. The establishment of Hs3st-B mutant and effective Hs3st-A RNAi lines provides essential tools for further studies of the physiological roles of Hs3st-A and Hs3st-B in development and homeostasis.     

Cell-autonomous heparanase modulates self-renewal and migration in bone marrow-derived mesenchymal stem cells

Background

Stem cell-fate is highly regulated by stem cell niche, which is composed of a distinct microenvironment, including neighboring cells, signals and extracellular matrix. Bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent stem cells and are potentially applicable in wide variety of pathological conditions. However, the niche microenvironment for BM-MSCs maintenance has not been clearly characterized. Accumulating evidence indicated that heparan sulfate glycosaminoglycans (HS-GAGs) modulate the self-renewal and differentiation of BM-MSCs, while overexpression of heparanase (HPSE1) resulted in the change of histological profile of bone marrow. Here, we inhibited the enzymatic activity of cell-autonomous HPSE1 in BM-MSCs to clarify the physiological role of HPSE1 in BM-MSCs.

Results

Isolated mouse BM-MSCs express HPSE1 as indicated by the existence of its mRNA and protein, which includes latent form and enzymatically active HPSE1. During in vitro osteo-differentiations, although the expression levels of Hpse1 fluctuated, enzymatic inhibition did not affect osteogenic differentiation, which might due to increased expression level of matrix metalloproteinase 9 (Mmp9). However, cell proliferation and colony formation efficiency were decreased when HPSE1 was enzymatically inhibited. HPSE1 inhibition potentiated SDF-1/CXCR4 signaling axis and in turn augmented the migratory/anchoring behavior of BM-MSCs. We further demonstrated that inhibition of HPSE1 decreased the accumulation of acetylation marks on histone H4 lysine residues suggesting that HPSE1 also modulates the chromatin remodeling.

Conclusions

Our findings indicated cell-autonomous HPSE1 modulates clonogenicity, proliferative potential and migration of BM-MSCs and suggested the HS-GAGs may contribute to the niche microenvironment of BM-MSCs.     

Hippocampal proteoglycans brevican and versican are linked to spatial memory of Sprague–Dawley rats in the morris water maze

Proteoglycans (PGs) are major constituents of the extracellular matrix and have recently been proposed to contribute to synaptic plasticity. Hippocampal PGs have not yet been studied or linked to memory. The aim of the study, therefore, was to isolate and characterize rat hippocampal PGs and determine their possible role in spatial memory. PGs were extracted from rat hippocampi by anion‐exchange chromatography and analyzed by nano LC‐MS/MS. Twenty male Sprague–Dawley rats were tested in the morris water maze. PGs agrin, amyloid beta A4 protein, brevican, glypican‐1, neurocan, phosphacan, syndecan‐4, and versican were identified in the hippocampi. Brevican and versican levels in the membrane fraction were higher in the trained group, correlating with the time spent in the target quadrant. α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate receptor GluR1 was co‐precipitated with brevican and versican. Levels for a receptor complex containing GluR1 was higher in trained while GluR2 and GluR3‐containing complex levels were higher in yoked rats. The findings provide information about the PGs present in the rat hippocampus, demonstrating that versican and brevican are linked to memory retrieval in the morris water maze and that PGs interact with α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate receptor GluR1, which is linked to memory retrieval.

     

Regulation of proteoglycan and hyaluronan synthesis by elevated level of intracellular cyclic adenosine monophosphate in peritubular cells from immature rat testis

The effects of an increase in intracellular cAMP concentration on proteoglycan (PG) synthesis by peritubular (PT) cells from immature rat testis were investigated. In the presence of dBcAMP for 72 h, the [³H]-hexosamine incorporation in secreted PG and in cellassociated PG was reduced, whereas [³⁵S]-sulfate radioactivity was enhanced in secreted PG and not affected in cell-associated PG. Cholera toxin and IBMX, known to generate high intracellular cAMP levels, induced similar changes. Cyclic AMP did not alter PG protein moiety synthesis but enhanced PG turnover. Cholera toxin and dBcAMP profoundly modified PG characteristics: (1) Apparent molecular weight of PG was increased. (2) This was due to an increase in glycosaminoglycans (heparan sulfate (HS) and chondroitin sulfate (CS)) length. (3) The number of glycosaminoglycan chains was presumably reduced. (4) Heparan sulfate and chondroitin sulfate chains of medium and cell layer-associated PG appeared oversulfated. (5) The pattern of cell layer associated PG was modified with a decrease in HSPG and a correlative increase in CSPG. Cholera toxin and dBcAMP also dramatically stimulated hyaluronan synthesis by possible phosphorylation induced activation of hyaluronan synthase(s).     

Proteinases and myocardial extracellular matrix turnover

Extracellular structural remodeling is the compensatory response of the tissue following pathological stage. Myocardial infarction, which leads to adverse remodeling, thinning of the ventricle wall, dilatation and heart failure, is one of the leading causes of death. Remodeling implies an alteration in the extracellular matrix and in the spatial orientation of cells and intracellular components. The extracellular matrix is responsible for cardiac cell alignment and myocardial structural integrity. Substances that break down the extracellular matrix, specialized proteinases as well as inhibitors of proteinases, appear to be normally balanced in maintaining the integrity of the myocardium. Myocardial infarction leads to an imbalance in proteinase/ antiproteinase activities causing alterations in the stability and integrity of the extracellular matrix and adverse tissue remodeling. To explore mechanisms involved in this process and, in particular, to focus on matrix metalloproteinases, their inhibitors, and activators, an understanding of proteinase and antiproteinase is needed. This review represents new and significant information regarding the role of activated matrix proteinases antiproteinases in remodeling. Such information will have a significant impact both on the understanding of the basic cell biology of extracellular matrix turnover, as well as on potential avenues for pharmacological approaches to the treatment of ischemic heart disease and failure.     

Changes in proteoglycans of cultured pig aortic smooth muscle cells during subculture

Summary Smooth muscle cells were cultured from pig aorta. Changes in both the growth and the properties of sulfated proteoglycans were observed during passage. The population doubling time during log phase growth was 34 h from Passages 3 to 7–8 but 20 h at the Passage 11, and the cell density at the stationary phase, was 86 000 and 136 000 cells/cm² at Passages 3 and 11, respectively. Structural characteristics of sulfated proteoglycans secreted into the medium were investigated after metabolic labeling with [³⁵S]-sulfate. Significant differences were observed with age in vitro: a) [³⁵S]proteoglycan complexes were in a greater amount at Passage 10 than at Passage 3; b) the hydrodynamic size of at least 45% of subunits and about 90% of monomers decreased with in vitro aging; c) this decrease in the size of proteoglycans was partly due to a decrease in the size of their glycanic chains; d) an increase of 15% in the proportion of dermatan sulfate was observed when cells were subjected to 10 passages. This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale (INSERM, U. 181) and the Fondation pour la Recherche Médicale.     

Chondroitinase ABC promotes axonal re-growth and behavior recovery in spinal cord injury

In spinal cord injury, the injury could trigger some inhibitory signal cascades to promote chondroitin sulfate proteoglycans (CSPGs), the structures of scar tissues, formation. CSPGs could limit axonal regeneration mainly through the glycosaminoglycan (GAG) chain in the lesion site were suggested. We hypothesized that the digestion of CSPGs by chondroitinase ABC (ChABC) might decrease the inhibitory effects of limiting axonal re-growth after spinal cord injury. We compared the digesting products of CSPGs such as 2B6 by ChABC with the untreated control group and found no immunostaining of 2B6 in control group. The smaller size scars of ChABC-treatment were observed via CS-56, a type of CSPGs, 8 weeks after transection by immunohistochemistry. The inhibitory effects of CSPGs withdraw GAGs following ChABC-treatment would reduce, and immunopositive GAP-43 newly outgrown fibers were identified. In the animal trials, ChABC-treatment could improve motor function through BBB locomotor's test and reduce limiting ability of scar tissues to promote axonal regeneration via changing the structure of CSPGs by immunohistochemistry with GAP-43.     

CSPGs在大鼠脊髓损伤后的表达及其与GFAP的关系

目的:观察脊髓损伤后CSPGs的表达及其与GFAP的关系。方法:成年雄性SD大鼠25只,随机分为对照组和损伤组,损伤组分脊髓挤压损伤后0h、72h、1w、4w组,运用免疫荧光双重染色方法观察CSPGs与GFAP的表达。结果:挤压伤后损伤部位的CSPGs和GFAP的表达均增高,但二者的变化趋势并不一样。其中CSPGs从损伤后表达开始增高,此后一直增加,并在1w至4w时逐渐稳定,主要分布逐渐集中于损伤部位;星形胶质细胞的免疫反应也逐渐增加,其分布逐渐集中于损伤区域的边缘,逐渐形成胶质瘢痕界膜。损伤1w至4w,损伤区域内几乎没有了星形胶质细胞表达,但仍留有大量的CSPGs。结论:早期抑制星形胶质细胞分泌CSPGs,可以防止在损伤部位沉积大量的CSPGs,从而减小其对再生纤维的抑制作用。     

Myotube morphology, and expression and distribution of collagen type I during normal and low score normal avian satellite cell myogenesis

Myogenic satellite cells are essential for postnatal muscle growth and the regeneration of muscle in response to injury. An understanding of how the extracellular matrix affects satellite cell activity, and the temporal and spatial expression of extracellular matrix macromolecules is largely unknown. In the avian genetic muscle weakness, low score normal (LSN), satellite cell proliferation and differentiation rates are significantly lower than that observed in normal chicken satellite cells, which may be attributed to a late embryonic increase in the expression of decorin. Satellite cell-derived morphological properties, collagen type I expression, and the spatial distribution of collagen type I were investigated during normal and LSN satellite cell proliferation and differentiation. These studies showed a decrease in LSN myotube length and the number of nuclei per myotube. Collagen type I expression was similar between the LSN and normal satellite cell cultures during the course of proliferation and differentiation. However, the spatial distribution of collagen type I was altered in the LSN cultures 48 h after the initiation of fusion. The LSN cultures exhibited a premature extracellular distribution of collagen type I compared to the normal satellite cells.