Gene expression profiling of human mesenchymal stem cells chemotactically induced with CXCL12

In situ tissue engineering is a promising approach in regenerative medicine, with the possibility that adult stem or progenitor cells will be guided chemotactically to a tissue defect and subsequently differentiate into the surrounding tissue type. Mesenchymal stem cells (MSC) represent attractive candidate cells. Chemokines such as CXCL12 (SDF-1α) chemoattract MSC, but little is known about the molecular processes involved in the chemotaxis and migration of MSC. In this study, MSC recruitment by CXCL12 was investigated by genome-wide microarray analysis. The dose-dependent migration potential of bone-marrow-derived MSC toward CXCL12 was measured in an in vitro assay, with a maximum being recorded at a concentration of 1,000 nM CXCL12. Microarray analysis of MSC stimulated with CXCL12 and non-stimulated controls showed 30 differentially expressed genes (24 induced and six repressed). Pathway analysis revealed 11 differentially expressed genes involved in cellular movement and cytokine-cytokine receptor interaction, including those for migratory inducers such as the chemokines CXCL8 and CCL26, the leukocyte inhibitory factor, secretogranin II, and prostaglandin endoperoxide synthase 2. These results were confirmed by real-time polymerase chain reaction for selected genes. The obtained data provide further insights into the molecular mechanisms involved in chemotactic processes in cell migration and designate CXCL12 as a promising candidate for in situ recruitment in regenerative therapies. Stefan Stich and Marion Haag contributed equally to this work. This study was supported by the Investitionsbank Berlin and the European Regional Development Fund (grant: 10128098), Deutsche Forschungsgemeinschaft (grant: DFG SI 569/7–1), and the Bundesministerium für Bildung und Forschung (Bioinside: 13N9817).     

Overexpression of CXCL12 chemokine up-regulates connexin and integrin expression in mesenchymal stem cells through PI3K/Akt pathway

Abstract

Mesenchymal stem cells offer several potential advantages over other types of stem cells for cardiac repair. Nevertheless, poor survival of donor cells is one of the major concerns that hampers a better prognosis. Integrins, which involved in cell/extracellular matrix (ECM) interaction and connexins (Cxs), with a dual role as an anti-apoptotic and gap-junctional protein, can effectively resolve this issue. CXCL12, a member of the chemokine CXC subfamily, may play a role in stem cell survival and proliferation. CXCL12 activates several signaling pathways in stem cells, particularly the survival kinase, PI3K/Akt, which is also an important mediator of integrins and Cxs. Based on these characteristics of CXCL12, we investigated the potential of CXCL12 overexpression to induce integrin and connexin expression via PI3K/Akt pathway. Mesenchymal stem cells were transfected with adenovirus for increasing CXCL12 secretion. Membranous integrin and Cx expression as well as Akt expression levels were evaluated using Western blot analysis. Transfection resulted in increased CXCL12 in situ. Increased CXCL12 elevated membrane Cx43, Cx45, and integrin αVβ3 expression, as well as Cx phosphorylaton, which was activated by PI3K/Akt pathway. This mechanism may serve to improve mesenchymal stem cell viability in host tissue.     

DNA (cytosine-5)-methyltransferase 1 as a mediator of mutant p53-determined p16<Superscript>ink4A</Superscript> down-regulation

In cancer, gene silencing via hypermethylation is as common as genetic mutations in p53. Understanding the relationship between mutant p53 and hypermethylation of other tumor suppressor genes is essential when elucidate mechanisms of tumor development. In this study, two isogenic human B lymphoblast cell lines with different p53 status include TK6 containing wild-type p53 and WTK1 with mutant p53 were used and contrasted. Lower levels of p16^ink4A protein were detected in WTK1 cells than in TK6 cells, which were accompanied by increased DNA (cytosine-5)-methyltransferase 1 (DNMT1) gene expression as well as hypermethylation of the p16 ^ink4A promoter. siRNA experiments to transiently knock down wild-type p53 in TK6 cells resulted in increase of DNMT1 expression as well as decrease of p16^ink4A protein. Conversely, siRNA knockdown of mutant p53 in WTK1 cells did not alter either DNMT1 or p16^ink4A protein levels. Furthermore, loss of suppression function of mutant p53 to DNMT1 in WTK1 was caused by the attenuation of its binding ability to the DNMT1 promoter. In summary, we provide evidences to elucidate the relationship between mutant p53 and DNMT1. Our results indicate that mutant p53 loses its ability to suppress DNMT1 expression, and thus enhances methylation levels of the p16 ^ink4A promoter and subsequently down-regulates p16^ink4A protein. Z. Guo and M.-H. Tsai contributed equally to this work.     

CXCL12 overexpression and secretion by aging fibroblasts enhance human prostate epithelial proliferation in vitro

The direct relationship between the aging process and the incidence and prevalence of both benign prostatic hyperplasia (BPH) and prostate cancer (PCa) implies that certain risk factors associated with the development of both diseases increase with the aging process. In particular, both diseases share an overly proliferative phenotype, suggesting that mechanisms that normally act to suppress cellular proliferation are disrupted or rendered dysfunctional as a consequence of the aging process. We propose that one such mechanism involves changes in the prostate microenvironment, which 'evolves' during the aging process and disrupts paracrine interactions between epithelial and associated stromal fibroblasts. We show that stromal fibroblasts isolated from the prostates of men 63-81 years of age at the time of surgery express and secrete higher levels of the CXCL12 chemokine compared with those isolated from younger men, and stimulate CXCR4-mediated signaling pathways that induce cellular proliferation. These studies represent an important first step towards a mechanistic elucidation of the role of aging in the etiology of benign and malignant prostatic diseases.     

Spatiotemporal expression and functional implication of CXCL14 in the developing mice cerebellum

Cerebellar granule neurons migrate from the external granule cell layer (EGL) to the internal granule cell layer (IGL) during postnatal morphogenesis. This migration process through 4 different layers is a complex mechanism which is highly regulated by many secreted proteins. Although chemokines are well-known peptides that trigger cell migration, but with the exception of CXCL12, which is responsible for prenatal EGL formation, their functions have not been thoroughly studied in granule cell migration. In the present study, we examined cerebellar CXCL14 expression in neonatal and adult mice. CXCL14 mRNA was expressed at high levels in adult mouse cerebellum, but the protein was not detected. Nevertheless, Western blotting analysis revealed transient expression of CXCL14 in the cerebellum in early postnatal days (P1, P8), prior to the completion of granule cell migration. Looking at the distribution of CXCL14 by immunohistochemistry revealed a strong immune reactivity at the level of the Purkinje cell layer and molecular layer which was absent in the adult cerebellum. In functional assays, CXCL14 stimulated transwell migration of cultured granule cells and enhanced the spreading rate of neurons from EGL microexplants. Taken together, these results revealed the transient expression of CXCL14 by Purkinje cells in the developing cerebellum and demonstrate the ability of the chemokine to stimulate granule cell migration, suggesting that it must be involved in the postnatal maturation of the cerebellum.     

趋化因子CXCL12及其受体CXCR4在胶质瘤中的研究进展

CXCL12是趋化因子家族成员之一,是能够特异性结合其受体CXCR4发挥趋化性作用的细胞因子。最初,CXCL12及CXCR4被发现于炎症细胞,参与机体炎症、免疫等病理反应。接下来的几年中发现,它在机体发育、成熟过程中也有重要作用。如今,大量研究表明它与肿瘤的生长、侵袭及转移密切相关。据报道,在乳腺癌、肺癌、卵巢癌等二十余种肿瘤组织中发现CXCL12及CXCR4的表达,其中也包括中枢系统肿瘤-胶质瘤。CXCL12/CXCR4参与胶质瘤生长过程的多个步骤,包括肿瘤增殖、侵袭、转移等。有实验指出,转移灶的CXCR4表达水平较原发灶高,CXCR4有可能成为抑制胶质瘤生长、转移的重要靶目标。     

Downregulating the expression of heparanase inhibits the invasion, angiogenesis and metastasis of human hepatocellular carcinoma

Invasion and metastasis are key features of human hepatocellular carcinoma (HCC). Heparanase is an endoglycosidase that can degrade extracellular matrix by cleaving heparan sulfate chains of heparan sulfate proteoglycan, thus playing important roles in the invasion and metastasis of human cancers. Heparanase has been detected in various human cancers and regarded as a prospective target in human cancer treatments. However, the effects of inhibiting the expression of heparanase on human HCC have not been fully evaluated. In this article we show that downregulating the expression of heparanase either by antisense oligodeoxynucleotide or by RNA interferencing can significantly reduce the expression of heparanase in SMMC7721 human HCC cells, leading to inhibition of the invasiveness, metastasis, and angiogenesis of HCC cells both in vitro and in vivo. Our results suggest that genetic downregulation of the expression of heparanase may serve as an efficient cancer therapeutic for human HCC.     

Stromal cell-derived factor-1alpha (SDF-1alpha/CXCL12) stimulates ovarian cancer cell growth through the EGF receptor transactivation

Ovarian cancer (OC) is the leading cause of death in gynecologic diseases in which there is evidence for a complex chemokine network. Chemokines are a family of proteins that play an important role in tumor progression influencing cell proliferation, angiogenic/angiostatic processes, cell migration and metastasis, and, finally, regulating the immune cells recruitment into the tumor mass. We previously demonstrated that astrocytes and glioblastoma cells express both the chemokine receptor CXCR4 and its ligand stromal cell-derived factor-1 (SDF-1), and that SDF-1alpha treatment induced cell proliferation, supporting the hypothesis that chemokines may play an important role in tumor cells' growth in vitro. In the present study, we report that CXCR4 and SDF-1 are expressed in OC cell lines. We demonstrate that SDF-1alpha induces a dose-dependent proliferation in OC cells, by the specific interaction with CXCR4 and a biphasic activation of ERK1/2 and Akt kinases. Our results further indicate that CXCR4 activation induces EGF receptor (EGFR) phosphorylation that in turn was linked to the downstream intracellular kinases activation, ERK1/2 and Akt. In addition, we provide evidence for cytoplasmic tyrosine kinase (c-Src) involvement in the SDF-1/CXCR4-EGFR transactivation. These results suggest a possible important "cross-talk" between SDF-1/CXCR4 and EGFR intracellular pathways that may link signals of cell proliferation in ovarian cancer.     

Glycosaminoglycans and syndecan-4 are involved in SDF-1/CXCL12-mediated invasion of human epitheloid carcinoma HeLa cells

Background

In addition to their physiologic effects in inflammation and angiogenesis, chemokines are involved in cancer pathology. The CXC-chemokine stromal cell-derived factor-1 (SDF-1)/CXCL12 mediates its biological activities through activation of G protein-coupled receptor CXCR4 and binds to glycosaminoglycans (GAGs).

Methods

Using Bio-coat cell migration chambers, specific antagonists, flow cytometry and RNA interference, we evaluate the involvement of heparan sulfate proteoglycans (HSPG) in the SDF-1/CXCL12-induced invasion of human cervix epitheloid carcinoma HeLa cells.

Results

The SDF-1/CXCL12-induced cell invasion is dependent on CXCR4. Furthermore, Protein Kinase C delta (PKC δ) and c-jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) are implicated in this event, but not extracellular signal-regulated kinase (ERK) 1/2. Moreover, the invasion of HeLa cells induced by SDF-1/CXCL12 was dependent on matrix metalloproteinase-9 (MMP-9). The pre-incubation of HeLa cells with heparin or with anti-heparan sulfate antibodies or with β-d-xyloside inhibited SDF-1/CXCL12-mediated cell invasion. Furthermore, the down-regulation of syndecan-4, a heparan sulfate proteoglycan, decreased SDF-1/CXCL12-mediated HeLa cell invasion. GAGs, probably on syndecan-4, are involved in SDF-1/CXCL12-mediated cell chemotaxis.

General significance

These data suggest that targeting the glycosaminoglycan/chemokine interaction could be a new therapeutic approach for carcinomas in which SDF-1/CXCL12 is involved.     

Identification of the suppressive factors for human immunodeficiency virus type-1 replication using the siRNA mini-library directed against host cellular genes

We performed the screening to find the novel host factors affecting human immunodeficiency virus type-1 (HIV-1) replication using the siRNA mini-library consisted with 257 siRNAs directed against cellular genes. J111 cells, a human acute monocytic leukemia cell line, were transfected with individual siRNA, followed by either infected or transfected with the HIV-1 molecular clone with luciferase reporter gene in 96-well plate format. The results showed that six siRNAs significantly enhanced the HIV-1 replication in J111 cells, indicating that the target cellular genes of those siRNAs may negatively regulate HIV-1 replication in normal cell culture condition. We also discuss the possible mechanisms by which those cellular proteins regulate viral replication.     

哺乳动物中RNA干扰沉默基因表达的研究进展

RNA干扰及相关基因沉默导致的代谢通路变化已经彻底改变了人们对基因调控的理解。基因沉默技术已经被用来作为一种研究工具来控制某些细胞基因的表达,特异的si RNA导入不同的细胞需要不同的转染载体才能达到最大的转染效率,并且不会产生较大的细胞毒性。近年来尤其是碳纳米管等新型纳米材料载体的应用拓展了人们对传统脂质体和病毒载体的认识。首先介绍RNA干扰技术及其发展历程,随后对利用几种不同的载体来设计和进行RNA干扰试验进行了比较,最后对最初的脂质体到生物病毒再到高分子纳米材料介导的RNA干扰在疾病的治疗和临床诊断进行了展望。