Mast Cell Tryptase Promotes Inflammatory Bowel Disease-Induced Intestinal Fibrosis.

BACKGROUND: Intestinal fibrosis is the final pathological outcome of chronic intestinal inflammation without specific therapeutic drugs, which leads to ileus and surgical intervention. Intestinal fibrosis is characterized by excessive deposition of extracellular matrix (ECM). The role of mast cells (MCs), which are members of the sentinel immune cell population, is unknown in intestinal fibrosis. METHODS: In this study, we analyzed changes in MCs, tryptase proteins, and ECM components in human fibrotic and control patient intestines. We constructed dextran sodium sulfate-induced intestinal fibrosis models using wild-type mice, MC-reconstituted mice, and MC-deficient mice to explore the role of MCs and tryptase in intestinal fibrosis. The roles and mechanisms of MCs and tryptase on fibroblasts were evaluated using human MCs (HMC-1 and LAD-2), commercial tryptase proteins, human colon fibroblasts (CCD-18Co fibroblasts), the tryptase inhibitor APC366, and the protease-activated receptor-2 (PAR-2) antagonist ENMD-1068. RESULTS: Regardless of whether the colon was a human colon or a mouse colon, the fibrotic intestinal tissue had increased MC infiltration and a higher expression of ECM proteins or genes than that of the control group. The dextran sodium sulfate-induced intestinal fibrosis in MC-deficient mice was alleviated compared with that in wild-type mice. After MC reconstruction in MC-deficient mice, the alleviating effect disappeared. Tryptase, as a content stored in MC granules, was released into fibrotic intestinal tissues in the form of degranulation, resulting in an increased expression of tryptase. Compared with the control group, the tryptase inhibition group (the APC366 group) had reduced intestinal fibrosis. The CCD-18Co fibroblasts, when cocultured with MCs or treated with tryptase proteins, were activated to differentiate into myofibroblasts and secrete more ECM proteins (such as collagen and fibronectin). The underlying mechanism of fibroblast activation by tryptase was the activation of the PAR-2/Akt/mTOR pathway. CONCLUSIONS: We found that MC tryptase promotes inflammatory bowel disease-induced intestinal fibrosis. The underlying mechanism is that tryptase promotes the differentiation of fibroblasts into fibrotic-phenotype myofibroblasts by activating the PAR-2/Akt/ mTOR pathway of fibroblasts.

Icariin Ameliorates Amyloid Pathologies by Maintaining Homeostasis of Autophagic Systems in Aß1-42-Injected Rats.

Macroautophagy, a sole pathway for dysfunctional organelles or aggregated proteins turnover, has been implicated in the early development of Alzheimer's disease (AD). Previous studies have found that reversal of autophagy dysfunction in APP transgenic mice ameliorates amyloid pathologies. Icariin (ICA), the main component from traditional Chinese herb Epimedium brevicornu Maxim., can reduce accumulations of amyloid-ß (Aß) peptide in vivo and in vitro, but the mechanism remains unclear. Here, we explored the effects of ICA on autophagy-lysosomal pathway in intracerebroventricular (icv) injection of human Aß1-42 peptide rats. We demonstrated that feeding the rats with ICA (30 mg/kg, 60 mg/kg and 90 mg/kg rat, per os) for 4 weeks rescued the Aß1-42-induced spatial memory impairments, reduced endogenous rat Aß42 tested by ELISA and decreased Aß accumulation using 6E10 antibody. Furthermore, Aß1-42 induced strong autophagy response, however ICA decreased the levels of microtubule-associated protein 1 light chain 3 (LC3) II/LC3I, Beclin1, Cathepsin D (Cat D) and brain lysosomal Cathepsin D activity. We also observed that ICA enhanced the phosphorylation of protein kinase B (PKB/AKT) and p70 ribosomal protein S6 kinase (p70S6K). In addition, ICA arrested Aß1-42-induced cells loss, mitochondrias damage, nuclear membranes unclear and abundant nucleas chromatin agglutinates in hippocampus, lessened the expression of Cleaved-caspase-3, brain oxidative stress, astroglial activation. These findings suggest that ICA can ameliorate amyloid pathologies with improving autophagy-lysosome function and Chinese materia medica may be potential for AD treatment.

NGF promotes long-term memory formation by activating poly(ADP-ribose)polymerase-1.

Nerve growth factor (NGF) is a critical secreted protein that plays an important role in development, survival, and function of the mammalian nervous system. Previously reports suggest that endogenous NGF is essential for the hippocampal plasticity/memory and NGF deprivation induces the impairment of hippocampus-related memory and synaptic plasticity. However, whether exogenous supplement of NGF could promote the hippocampus-dependent synaptic plasticity/memory and the possible underlying mechanisms are not clear. In this study we found that NGF administration facilitates the hippocampus-dependent long-term memory and synaptic plasticity by increasing the activity of PARP-1, a polymerase mediating the PolyADP-ribosylation and important for the memory formation. Co-application of 3-Aminobenzamide (3-AB), a specific inhibitor of PARP-1, distinctly blocked the boosting effect of NGF on memory and synaptic plasticity, and the activation of downstream PKA-CREB signal pathway. Our data provide the first evidence that NGF supplement facilitates synaptic plasticity and the memory ability through PARP-1-mediated protein polyADP-ribosylation and activation of PKA-CREB pathway.

Glycogen synthase kinase-3ß regulates leucine-309 demethylation of protein phosphatase-2A via PPMT1 and PME-1.

Protein phosphatase-2A (PP2A) activity is significantly suppressed in Alzheimer's disease. We have reported that glycogen synthase kinase-3ß (GSK-3ß) inhibits PP2A via upregulating the phosphorylation of PP2A catalytic subunit (PP2A(C)). Here we studied the effects of GSK-3ß on the inhibitory demethylation of PP2A at leucine-309 (dmL309-PP2A(C)). We found that GSK-3ß regulates dmL309-PP2A(C) level by regulating PME-1 and PPMT1. Knockdown of PME-1 or PPMT1 eliminated the effects of GSK-3ß on PP2A(C). GSK-3 could negatively regulate PP2A regulatory subunit protein level. We conclude that GSK-3ß can inhibit PP2A by increasing the inhibitory L309-demethylation involving upregulation of PME-1 and inhibition of PPMT1.

Glycogen synthase kinase-3ß regulates Tyr307 phosphorylation of protein phosphatase-2A via protein tyrosine phosphatase 1B but not Src.

GSK-3ß (glycogen synthase kinase-3ß), a crucial tau kinase, negatively regulates PP2A (protein phosphatase 2A), the most active tau phosphatase that is suppressed in the brain in AD (Alzheimer's disease). However, the molecular mechanism is not understood. In the present study we found that activation of GSK-3ß stimulates the inhibitory phosphorylation of PP2A at Tyr307 (pY307-PP2A), whereas inhibition of GSK-3ß decreased the level of pY307-PP2A both in vitro and in vivo. GSK-3ß is a serine/threonine kinase that can not phosphorylate tyrosine directly, therefore we measured PTP1B (protein tyrosine phosphatase 1B) and Src (a tyrosine kinase) activities. We found that GSK-3ß can modulate both PTP1B and Src protein levels, but it only inhibits PTP1B activity, with no effect on Src. Furthermore, only knockdown of PTP1B but not Src by siRNA (small interfering RNA) eliminates the effects of GSK-3ß on PP2A. GSK-3ß phosphorylates PTP1B at serine residues, and activation of GSK-3ß reduces the mRNA level of PTP1B. Additionally, we also observed that GSK-3 negatively regulates the protein and mRNA levels of PP2A, and knockdown of CREB (cAMP-response-element-binding protein) abolishes the increase in PP2A induced by GSK-3 inhibition. The results of the present study suggest that GSK-3ß inhibits PP2A by increasing the inhibitory Tyr307 phosphorylation and decreasing the expression of PP2A, and the mechanism involves inhibition of PTP1B and CREB.

Acetyl-L-carnitine attenuates okadaic acid induced tau hyperphosphorylation and spatial memory impairment in rats.

Tau hyperphosphorylation and memory deficit are characteristic alterations of Alzheimer's disease (AD). Protein phosphatases (PP) 2A plays a crucial role in AD-like lesions. Inhibition of PP2A through hippocampal injection of okadaic acid (OA) induces tau hyperphosphorylation and memory impairment of rats. By using this model, we explored in the present study the effects of acetyl-L-carnitine (ALCAR), a constituent of the inner mitochondrial membrane, on the memory retention, tau phosphorylation, and oxidative stress in rats. We found that pre-treatment of ALCAR (50 mg/d . rat, per os) for two weeks efficiently improved the OA-induced spatial memory retention impairment of the rats. ALCAR antagonized tau hyperphosphorylation at multiple AD sites and it abated the OA-induced PP2A inhibition and oxidative stress. Our study provides the first in vivo evidence that ALCAR can attenuate AD-like PP2A inhibition, tau hyperphosphorylation, and spatial memory deficit of the rats. It suggests that ALCAR may hold potential in AD treatment.

Activation of glycogen synthase kinase-3 inhibits long-term potentiation with synapse-associated impairments.

Activation of glycogen synthase kinase-3 (GSK-3) can cause memory deficits as seen in Alzheimer's disease, the most common age-associated dementia, but the mechanism is not understood. Here, we found that activation of GSK-3 by wortmannin or transient overexpression of wild-type GSK-3beta could suppress the induction of long-term potentiation (LTP) in rat hippocampus, whereas simultaneous inhibition of GSK-3 by lithium or SB216763 or transient expression of a dominant-negative GSK-3beta mutant (dnGSK-3beta) preserved the LTP. After high-frequency stimulation (HFS), the presynaptic release of glutamate and the expression/clustering of synapsin I, a synaptic vesicle protein playing an important role in neurotransmitter release, decreased markedly after upregulation of GSK-3. In vitro studies further demonstrated that GSK-3 inhibited the expression of SynI independent of HFS. In postsynaptic level, the expression of PSD93 and NR2A/B proteins decreased significantly when GSK-3 was activated. The LTP-associated synapse impairments including less presynaptic active zone, thinner postsynaptic density, and broader synaptic cleft were also prominent in the hippocampal slices after HFS with activation of GSK-3. These synaptic impairments were attenuated when GSK-3 was simultaneously inhibited by LiCl or SB216763 or transient expression of dnGSK-3. We conclude that upregulation of GSK-3 impairs the synaptic plasticity both functionally and structurally, which may underlie the GSK-3-involved memory deficits.