Kallikrein Transduced Mesenchymal Stem Cells Protect against Anti-GBM Disease and Lupus Nephritis by Ameliorating Inflammation and Oxidative Stress

Previously we have shown that kallikreins (klks) play a renoprotective role in nephrotoxic serum induced nephritis. In this study, we have used mesenchymal stem cells (MSCs) as vehicles to deliver klks into the injured kidneys and have measured their therapeutic effect on experimental antibody induced nephritis and lupus nephritis. Human KLK-1 (hKLK1) gene was transduced into murine MSCs using a retroviral vector to generate a stable cell line, hKLK1-MSC, expressing high levels of hKLK1. 129/svj mice subjected to anti-GBM induced nephritis were transplanted with 10⁶ hKLK1-MSCs and hKLK1 expression was confirmed in the kidneys. Compared with vector-MSCs injected mice, the hKLK1-MSCs treated mice showed significantly reduced proteinuria, blood urea nitrogen (BUN) and ameliorated renal pathology. Using the same strategy, we treated lupus-prone B6.Sle1.Sle3 bicongenic mice with hKLK1-MSCs and demonstrated that hKLK1-MSCs delivery also attenuated lupus nephritis. Mechanistically, hKLK1-MSCs reduced macrophage and T-lymphocyte infiltration into the kidney by suppressing the expression of inflammation cytokines. Moreover, hKLK1 transduced MSCs were more resistant to oxidative stress-induced apoptosis. These findings advance genetically modified MSCs as potential gene delivery tools for targeting therapeutic agents to the kidneys in order to modulate inflammation and oxidative stress in lupus nephritis.     

Long-term D-galactose injection combined with ovariectomy serves as a new rodent model for Alzheimer's disease

Estrogen deprivation and oxidative stress have been well established as two main factors closely related to the pathological development of Alzheimer's disease (AD). The aim of the present study is to investigate whether these two components act synergistically to accelerate the pathophysiological course of AD. To do this, we examined the effect of long-term intraperitoneal administration of D-galactose (D-gal) into ovariectomized (OVX) rats. Six weeks later, the OVX and d-gal-injected rats exhibited a higher degree of cognitive and memory impairment. This was accompanied by cholinergic neuronal loss in the forebrain and synaptic degeneration in the hippocampus and cerebral cortex which was not observed in intact controls, animals receiving injections of d-gal alone, untreated OVX animals or OVX animals receiving both D-gal and 17-beta estradiol. The typical histopathological alterations associated with AD, including intracellular deposition of amyloid beta peptide and the appearance of intracellular neurofibrillary tangles and nuclear granulovacuolar bodies, were observed in the hippocampus of OVX and D-gal-injected rats but not in other control groups. These results strongly suggest that estrogen deprivation and oxidative stress behave synergistically to enhance the development and progression of AD. Long-term OVX combined with D-gal injection serves as an ideal AD rodent model capable of mimicking pathological, neurochemical and behavioral alterations in AD.     

Profiling the DNA-binding specificities of engineered Cys2His2 zinc finger domains using a rapid cell-based method

The C₂H₂ zinc finger is the most commonly utilized framework for engineering DNA-binding domains with novel specificities. Many different selection strategies have been developed to identify individual fingers that possess a particular DNA-binding specificity from a randomized library. In these experiments, each finger is selected in the context of a constant finger framework that ensures the identification of clones with a desired specificity by properly positioning the randomized finger on the DNA template. Following a successful selection, multiple zinc-finger clones are typically recovered that share similarities in the sequences of their DNA-recognition helices. In principle, each of the clones isolated from a selection is a candidate for assembly into a larger multi-finger protein, but to date a high-throughput method for identifying the most specific candidates for incorporation into a final multi-finger protein has not been available. Here we describe the development of a specificity profiling system that facilitates rapid and inexpensive characterization of engineered zinc-finger modules. Moreover, we demonstrate that specificity data collected using this system can be employed to rationally design zinc fingers with improved DNA-binding specificities.     

Role of the growth hormone (GH) receptor transmembrane domain in receptor predimerization and GH-induced activation

The GH receptor (GHR) mediates GH effects by activating the GHR-associated cytoplasmic tyrosine kinase, Janus kinase 2. Recent studies indicate that GHRs exist as dimers independently of GH binding. Some authors suggest that receptor predimerization is mediated by the transmembrane domain (TMD) and that GH binding initiates signaling by triggering changes in the orientation of the two GHRs within the dimer. In this study, we investigate the role of GHR TMD in GH-independent receptor dimerization and ligand-induced activation. We prepared a GHR mutant, GHR(LDLR), in which the TMD is replaced with the TMD of the human low-density lipoprotein receptor (LDLR). The resultant chimera has a TMD two residues shorter than the native GHR TMD; thus, in addition to possessing a different TMD, the altered GHR(LDLR) TMD helical register may change positions of the GHR extracellular domain (ECD) and intracellular domain relative to the TMD when compared with the wild-type (WT) receptor. When each was coexpressed with an intracellular domain-truncated GHR mutant, GHR(1-274-Myc), both WT GHR and GHR(LDLR) were specifically coprecipitated with GHR(1-274-Myc), indicating that the GHR TMD was not required for GHR heterodimerization with GHR(1-274-Myc). We further examined the contribution of the so-called "dimerization interface," a GHR ECD region that is critical for GH-induced signaling, to receptor predimerization. Coimmunoprecipitation experiments with either WT GHR, a dimerization interface mutant (GHR-H150D), or a control mutant (GHR-T147D) with GHR(1-274-Myc) showed dramatically reduced coprecipitation of GHR-H150D with GHR(1-274-Myc) when compared with WT GHR or GHR-T147K. This result suggests that, in contrast to some recent models, the dimerization interface contributes to GHR predimerization. We also compared WT GHR with GHR(LDLR) and GHR(LDLRDelta4) (a chimera in which the LDLR TMD has an internal deletion of four residues) with regard to response to GH stimulation. Although the chimeras had similar GH dose responses and time courses for signaling as WT GHR, they were markedly less sensitive to inhibition of signaling by a conformation-sensitive GHR ECD monoclonal antibody. Further, the chimeras were much less sensitive to inducible metalloprotease cleavage than was WT GHR, implying that the ECD conformations of the chimera receptors differ from WT GHR. Collectively, our data indicate that the composition and/or length of the TMD affect some aspects of GHR function, but do not affect receptor predimerization or GH-induced GHR activation. Further, they suggest that the GHR ECD-TMD is more flexible than previously thought in terms of the ability to achieve the active conformation in response to GH.     

The role of EGF‐EGFR signalling pathway in hepatocellular carcinoma inflammatory microenvironment

Epidermal growth factor (EGF) and their receptor (EGFR) play an important role in the development of cancer proliferation, and metastasis, although the mechanism remains unclear. The present study aimed at investigating the role of EGF‐EGFR signalling pathway in the development of human hepatocellular carcinoma (HCC) inflammatory environment. Gene profiles of inflammatory cytokines from HCC were measured. Cell bio‐behaviours of HCC with low or high metastasis were detected by the live cell monitoring system. Cell proliferation was measured by CCK8. The protein level of CXCL5 and CXCL8 was measured by ELISA. The phosphorylation of PI3K, ERK, MAPK was measured by western blot. EGF significantly induced cell proliferation in HepG2 cells, but not in HCCLM3 cells. EGF prompted the cell movement in both HepG2 and HCCLM3 and regulated the production of CXCL5 and CXCL8 from HCC, which were inhibited by EGFR inhibitor, Erk inhibitor (U0126), or PI3K inhibitors (BEZ‐235 and SHBM1009). HCC proliferation, metastasis and production of inflammatory cytokines were regulated via EGF‐EGFR signal pathways. CXCL5 could interact with CXCL8, possibly by CXCR2 or the cross‐talk between CXCR2 and EGFR. EGF‐EGFR signaling pathway can be the potential target of therapies for HCC.     

Variations of chromosomes 2 and 3 gene expression profiles among pulmonary telocytes,pneumocytes, airway cells,mesenchymal stem cells and lymphocytes

Telocytes (TCs) were identified as a distinct cellular type of the interstitial tissue and defined as cells with extremely long telopodes (Tps). Our previous data demonstrated patterns of mouse TC‐specific gene profiles on chromosome 1. The present study focuses on the identification of characters and patterns of TC‐specific or TC‐dominated gene expression profiles in chromosome 2 and 3, the network of principle genes and potential functional association. We compared gene expression profiles of pulmonary TCs, mesenchymal stem cells, fibroblasts, alveolar type II cells, airway basal cells, proximal airway cells, CD8⁺T cells from bronchial lymph nodes (T‐BL), and CD8⁺ T cells from lungs (T‐LL). We identified that 26 or 80 genes of TCs in chromosome 2 and 13 or 59 genes of TCs up‐ or down‐regulated in chromosome 3, as compared with other cells respectively. Obvious overexpression of Myl9 in chromosome 2 of TCs different from other cells, indicates that biological functions of TCs are mainly associated with tissue/organ injury and ageing, while down‐expression of Pltp implies that TCs may be associated with inhibition or reduction of inflammation in the lung. Dominant overexpression of Sh3glb1, Tm4sf1 or Csf1 in chromosome 3 of TCs is mainly associated with tumour promotion in lung cancer, while most down‐expression of Pde5 may be involved in the development of pulmonary fibrosis and other acute and chronic interstitial lung disease.     

Cytosolic chloride ion is a key factor in lysosomal acidification and function of autophagy in human gastric cancer cell

The purpose of the present study was to clarify roles of cytosolic chloride ion (Cl⁻) in regulation of lysosomal acidification [intra-lysosomal pH (pH_lys)] and autophagy function in human gastric cancer cell line (MKN28). The MKN28 cells cultured under a low Cl⁻ condition elevated pH_lys and reduced the intra-lysosomal Cl⁻ concentration ([Cl⁻]_lys) via reduction of cytosolic Cl⁻ concentration ([Cl⁻]_c), showing abnormal accumulation of LC3II and p62 participating in autophagy function (dysfunction of autophagy) accompanied by inhibition of cell proliferation via G₀/G₁ arrest without induction of apoptosis. We also studied effects of direct modification of H⁺ transport on lysosomal acidification and autophagy. Application of bafilomycin A1 (an inhibitor of V-type H⁺-ATPase) or ethyl isopropyl amiloride [EIPA; an inhibitor of Na⁺/H⁺ exchanger (NHE)] elevated pH_lys and decreased [Cl⁻]_lys associated with inhibition of cell proliferation via induction of G₀/G₁ arrest similar to the culture under a low Cl⁻ condition. However, unlike low Cl⁻ condition, application of the compound, bafilomycin A1 or EIPA, induced apoptosis associated with increases in caspase 3 and 9 without large reduction in [Cl⁻]_c compared with low Cl⁻ condition. These observations suggest that the lowered [Cl⁻]_c primarily causes dysfunction of autophagy without apoptosis via dysfunction of lysosome induced by disturbance of intra-lysosomal acidification. This is the first study showing that cytosolic Cl⁻ is a key factor of lysosome acidification and autophagy.     

PLIN1 deficiency affects testicular gene expression at the meiotic stage in the first wave of spermatogenesis

PLIN1, a lipid droplet associated protein, has been implicated in playing a key role in the regulation of lipolysis and lipid storage in adipocytes. PLIN1 is found to be highly expressed in Leydig cells of testis, suggesting a potential role in steroidogenesis and spermatogenesis. In this study, we showed that PLIN1 was expressed in testis and that its mRNA levels declined significantly with development. To investigate the role of PLIN1, we take advantage of PLIN1-null mice. We found that the number of seminiferous tubules containing round spermatids was significantly increased at P21 (postnatal day 21). Furthermore, microarray analysis showed that there were 538 differentially expressed genes between PLIN1-null and wild-type mice at P21. The up-regulated genes in knockout mice were enriched in spermatogenesis by Gene Ontology classification. Among them, Prm1 and Wbp2nl are important for spermatogenesis which were confirmed by real-time PCR. Unexpectedly, the levels of serum testosterone and serum 17β-estradiol as well as steroidogenic genes are not altered in the PLIN1-null mice. Compared to the wild-type mice, no significant difference of fertility was found in the PLIN1-null mice. Therefore, these findings indicated that PLIN1 disruption leads to the increase of round spermatid-containing seminiferous tubules at the meiotic stage of the first wave of spermatogenesis through regulating spermatogenic related genes.