Ganoderma lucidum polysaccharides (GLPS) have been used as traditional Chinese medicine for their properties of cancer prevention and immunomodulation. However, it is unclear whether GLPS has therapeutic effect on anti-hepatocellular carcinoma (HCC) in vivo. In this study, the effect of GLPS and their impact on the balance of regulatory T cell (Treg) and effector T cell (Teff) was measured in a model of hepatoma-bearing mice. The effect of GLPS and their impact on the balance of regulatory T cell (Treg) and effector T cell (Teff) were measured in a model of hepatoma-bearing mice. Real-time PCR detected the levels of MicroRNAs (miRNAs) and mRNA. The effects of Tregs on Teff proliferation were determined via suppression assay. The mircroRNA-125b (miR-125b) inhibitor was used to down-regulate miR-125b expression. GLPS significantly suppressed tumor growth in hepatoma-bearing mice associated with an increase of the ratio of Teffs to Tregs. Moreover, GLPS eliminate Treg suppression of Teff proliferation with an increase in IL-2 secretion. Addition of GLPS to treat T cells inhibited Notch1 and FoxP3 expression through increase of miR-125b expression. In hepatoma-bearing mice, miR-125b inhibitor obviously abolished the effect of GLPS on tumor growth. This finding provides the novel evidence for GLPS on inhibition of HCC through miR-125b inhibiting Tregs accumulation and function.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP/protein kinase A (PKA)–regulated chloride channel whose phosphorylation controls anion secretion across epithelial cell apical membranes. We examined the hypothesis that cAMP/PKA stimulation regulates CFTR biogenesis posttranslationally, based on predicted 14-3-3 binding motifs within CFTR and forskolin-induced CFTR expression. The 14-3-3β, γ, and ε isoforms were expressed in airway cells and interacted with CFTR in coimmunoprecipitation assays. Forskolin stimulation (15 min) increased 14-3-3β and ε binding to immature and mature CFTR (bands B and C), and 14-3-3 overexpression increased CFTR bands B and C and cell surface band C. In pulse-chase experiments, 14-3-3β increased the synthesis of immature CFTR, reduced its degradation rate, and increased conversion of immature to mature CFTR. Conversely, 14-3-3β knockdown decreased CFTR B and C bands (70 and 55%) and elicited parallel reductions in cell surface CFTR and forskolin-stimulated anion efflux. In vitro, 14-3-3β interacted with the CFTR regulatory region, and by nuclear magnetic resonance analysis, this interaction occurred at known PKA phosphorylated sites. In coimmunoprecipitation assays, forskolin stimulated the CFTR/14-3-3β interaction while reducing CFTR's interaction with coat protein complex 1 (COP1). Thus 14-3-3 binding to phosphorylated CFTR augments its biogenesis by reducing retrograde retrieval of CFTR to the endoplasmic reticulum. This mechanism permits cAMP/PKA stimulation to make more CFTR available for anion secretion.

Apoptosis of renal tubular epithelial cells is a key feature of the pathogenicity associated with tubulointerstitial fibrosis and other kidney diseases. One factor that regulates important cellular processes like apoptosis and cell proliferation is HRD1, an E3 ubiquitin ligase that acts by promoting ubiquitylation and degradation of its target protein. However, the detailed mechanisms by which HRD1 acts as a regulator of apoptosis in renal tubular epithelial cells have not been established. In our previous liquid chromatography-tandem mass spectrometry (LC-MS/MS) study (Mol Endocrinol. 2016;30:600–613), we demonstrated that one substrate of HRD1 was eIF2α, a critical protein in the PERK-eIF2α-ATF4-CHOP signaling pathway of endoplasmic reticulum (ER) stress. Here, we show that eIF2α expression was increased and HRD1 expression decreased when apoptosis was induced in HKC-8 cells by palmitic acid (PA) or high glucose (HG). HRD1 expression was also lower in kidney tissues from mice with diabetic nephropathy (DN) than in control mice. Forced expression of HRD1 also inhibited apoptosis in HKC-8 cells, while HRD1 overexpression decreased the expression of phosphorylated eIF2α and eIF2α. Further analysis indicated that HRD1 interacted with eIF2α and promoted its ubiquitylation and degradation by the proteasome. Moreover, the HRD1 protection of PA-treated HKC-8 cells was blunted by transfection with Myc-eIF2α. Thus, eIF2α ubiquitylation by HRD1 protects tubular epithelial cells from apoptosis caused by HG and PA, indicating a novel upstream target for therapeutic prevention of renal tubulointerstitial injury.

Article published by the Endocrine Society Article published by the Endocrine Society

Recent studies have indicated that the development of acute and chronic kidney disease including renal fibrosis is associated with endoplasmic reticulum (ER) stress. S100 calcium-binding protein 16 (S100A16) as a novel member of the S100 family is involved in kidney disease; however, few studies have examined fibrotic kidneys for a relationship between S100A16 and ER stress. In our previous study, we identified GRP78 as a protein partner of S100A16 in HK-2 cells. Here, we confirmed a physical interaction between GRP78 and S100A16 in HK-2 cells and a markedly increased expression of GRP78 in the kidneys of unilateral ureteral occlusion mice. S100A16 overexpression in HK-2 cells by infection with Lenti-S100A16 also induced upregulation of ER stress markers, including GRP78, p-IRE1α, and XBP1s. Immunofluorescence staining demonstrated that the interaction between S100A16 and GRP78 predominantly occurred in the ER of control HK-2 cells. By contrast, HK-2 cells overexpressing S100A16 showed colocalization of S100A16 and GRP78 mainly in the cytoplasm. Pretreatment with BAPTA-AM, a calcium chelator, blunted the upregulation of renal fibrosis genes and ER stress markers induced by S100A16 overexpression in HK-2 cells and suppressed the cytoplasmic colocalization of GRP78 and S100A16. Co-immunoprecipitation studies suggested a competitive binding between S100A16 and IRE1α with GRP78 in HK-2 cells. Taken together, our findings demonstrate a significant increase in S100A16 expression in the cytoplasm following renal injury. GRP78 then moves into the cytoplasm and binds with S100A16 to promote the release of IRE1α. The subsequent phosphorylation of IRE1α then leads to XBP1 splicing that activates ER stress.