Current Molecular Medicine - Volume 12, Issue 2, 2012

After a decade of successful journey, Current Molecular Medicine welcomes its prospective growth in a new decade. In marching into the second year of my editorship, great efforts are continuously devoted to publication of high quality research articles dealing with mechanisms and therapies of various human diseases. In this issue, a total of 9 research articles have been selected. Article one is by Das's group reporting KLF2-mediated transcriptional regulation of arthritis via modulation of monocyte differentiation and function. These authors have previously demonstrated that Kruppel-like factor 2 (KLF2) inhibits proinflammatory activation of monocytes. In the current study, they show that significantly greater numbers of inflammatory subset of CD11b'F4/80'Ly6C' monocytes were recruited to the inflammatory sites in KLF2 hemizygous mice compared to the wild type littermate controls. In parallel, inflammatory mediators, MCP-1, Cox-2 and PAI-1 were significantly up-regulated in bone marrow-derived monocytes isolated from KLF2 hemizygous mice, in comparison to wild-type controls. Furthermore, they found that monocytes isolated from KLF2 hemizygous mice contained more matured and differentiated cells towards osteoclastic lineage, potentially contributing to the severity of cartilage and bone damage in induced arthritic mice. The severity of arthritis was also associated with enhanced expression of HSP60, HSP90 and MMP13, but attenuated expression of pPTEN, p21, p38 and HSP25/27 in bone marrow cells of arthritic KLF2 hemizygous mice compared to wild type littermate controls. These data provide new insights and evidences of KLF2-mediated transcriptional regulation of arthritis. The generation of functional neural progenitor cells (NPCs) independent of donor brain tissue and embryonic tissues is of great therapeutic interest with regard to regenerative medicine and the possible treatment of neurodegenerative disorders. Traditionally, NPCs are derived through the differentiation of embryonic stem cells (ESCs) and Induced pluripotent stem cells (iPSCs). However, the induction of NPCs from ESCs and iPSCs is a complicated process that increases the risk of neoplasia and undesired cell types. This process can be circumvented through the direct conversion of somatic cells from one cell type to another by ectopic expression of specifically defined transcription factors. Using gene expression profiling and parental cells from E/Nestin:EGFP transgenic mice as a monitoring system, Zheng's group in article 2, tested nine factors with the potential to directly convert fibroblasts into NPCs. They found that five of these factors can directly convert adult dermal fibroblasts into NPC-like cells (iNPCs), and the resulting iNPCs possessed similar properties as primary NPCs including proliferation, self-renewal and differentiation. Significantly, iNPCs also exhibit chemotactic properties similar to those of primary NPCs. Their results provide an important alternative strategy to generate iNPCs for cell replacement therapy of neurodegenerative diseases. Intraneuronal accumulation of abnormal phosphorylated tau (p-tau) is a molecular pathology in many neurodegenerative tauopathies, including Alzheimer's disease (AD) and frontotemporal dementia with parkinsonism-linked to chromosome 17 (FTDP-17). However, the underlying mechanism remains unclear. In article 3, Shen's group showed an inverse relationship between endoplasmic reticulum membrane ubiquitin ligase (E3) Hrd1 expression and p-tau accumulation in the hippocampal neurons of AD, and proposed that Hrd1 may be a negative regulator of p-tau. This study provides important insight into the molecular mechanisms of human tauopathies. Cancer metastasis is the main cause of mortality. How metastatic cells arising from primary tumors and targeting to specific organs remain largely unknown. One of the cytokine receptors, cysteine x cysteine (CXC) chemokine receptor 4 (CXCR4), initially linked with leukocyte trafficking, is found overexpressed in various tumors and mediates homing of tumor cells to distant sites expressing its cognate ligand CXCL12. Thus, identification of CXCR4 inhibitors has great potential to abrogate tumor metastasis. In article 4, the study by Liu and Pang's group demonstrated that xanthohumol (XN), a prenylflavonoid derived from the female flowers of the hops plant (Humulus lupulus. L), suppressed CXCR4 expression in various cancer cell types in a concentration- and time-dependent manner. They demonstrated that XN abolished cell invasion induced by CXCL12 in both breast and colon cancer cells. Mechanistically, XN could block endogenous activation of nuclear factor kappa B, a key transcription factor regulating the expression of CXCR4 in cancer cells. Together, their results suggested that XN, as a novel inhibitor of CXCR4, could be a promising therapeutic agent contributed to cancer treatment.....

Kruppel-like factor 2 (KLF2) plays an important role in the regulation of a variety of immune cells, including monocytes. We have previously shown that KLF2 inhibits proinflammatory activation of monocytes. However, the role of KLF2 in arthritis is yet to be investigated. In the current study, we show that recruitment of significantly greater numbers of inflammatory subset of CD11b+F4/80+Ly6C+ monocytes to the inflammatory sites in KLF2 hemizygous mice compared to the wild type littermate controls. In parallel, inflammatory mediators, MCP-1, Cox-2 and PAI-1 were significantly up-regulated in bone marrow-derived monocytes isolated from KLF2 hemizygous mice, in comparison to wild-type controls. Methylated-BSA and IL-1β-induced arthritis was more severe in KLF2 hemizygous mice as compared to the littermate wild type controls. Consistent with this observation, monocytes isolated from KLF2 hemizygous mice showed an increased number of cells matured and differentiated towards osteoclastic lineage, potentially contributing to the severity of cartilage and bone damage in induced arthritic mice. The severity of arthritis was associated with the higher expression of proteins such as HSP60, HSP90 and MMP13 and attenuated levels of pPTEN, p21, p38 and HSP25/27 molecules in bone marrow cells of arthritic KLF2 hemizygous mice compared to littermate wild type controls. The data provide new insights and evidences of KLF2-mediated transcriptional regulation of arthritis via modulation of monocyte differentiation and function.

The generation of functional neural progenitor cells (NPCs) independent of donor brain tissue and embryonic tissues is of great therapeutic interest with regard to regenerative medicine and the possible treatment of neurodegenerative disorders. Traditionally, NPCs are derived through the differentiation of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). However, the induction of NPCs from ESCs and iPSCs is a complicated process that increases the risk of neoplasia and undesired cell types. This process can be circumvented through the direct conversion of somatic cells from one cell type to another by ectopic expression of specifically defined transcription factors. Using gene expression profiling and parental cells from E/Nestin:EGFP transgenic mice as a monitoring system, we tested nine factors with the potential to directly convert fibroblasts into NPCs. We found that five of these factors can directly convert adult dermal fibroblasts into NPC-like cells (iNPCs), and the resulting iNPCs possessed similar properties as primary NPCs including proliferation, self-renewal and differentiation. Significantly, iNPCs also exhibit chemotactic properties similar to those of primary NPCs. These provide an important alternative strategy to generate iNPCs for cell replacement therapy of neurodegenerative diseases.

Intraneuronal accumulation of abnormal phosphorylated tau (p-tau) is a molecular pathology in many neurodegenerative tauopathies, including Alzheimer's disease (AD) and frontotemporal dementia with parkinsonism-linked to chromosome 17 (FTDP-17). However, the underlying mechanism remains unclear. Here, we showed an inverse relationship between endoplasmic reticulum membrane ubiquitin ligase (E3) Hrd1 expression and p-tau accumulation in the hippocampal neurons of AD, and proposed that Hrd1 may be a negative regulator of p-tau. This notion was further supported by in vitro study demonstrating that Hrd1 interacted with tau and promoted the degradation of total tau and p-tau as well. The degradation of tau depended on its Hrd1 E3 activity. Knockdown of endogenous Hrd1 with siRNA stabilized tau levels. In addition, inhibition of proteasome maintained tau level and increased Hrd1-mediated tau ubiquitination, suggesting the proteasome was involved in tau/p-tau degradation. Over-expression of Hrd1 significantly alleviated tau cytotoxicity and promoted cell survival. These results indicated that Hrd1 functions as an E3 targeting tau or abnormal p-tau for proteasome degradation. The study provides an important insight into the molecular mechanisms of human tauopathies.

Cancer metastasis is the main cause of death (90%), and only recently we have gained some insight into the mechanisms by which metastatic cells arise from primary tumors and target to specific organs. Cysteine X Cysteine (CXC) chemokine receptor 4 (CXCR4), initially linked with leukocyte trafficking, is overexpressed in various tumors and mediates homing of tumor cells to distant sites expressing its cognate ligand CXCL12. Therefore, identification of CXCR4 inhibitors has great potential to abrogate tumor metastasis. In this study, we demonstrated that xanthohumol (XN), a prenylflavonoid derived from the female flowers of the hops plant (Humulus lupulus. L), suppressed CXCR4 expression in various cancer cell types in a concentration- and time-dependent manner. Both proteasome and lysosomal inhibitors had no effect to prevent the XN-induced downregulation of CXCR4, suggesting that the inhibitory effect of XN was not due to proteolytic degradation but occurred at the transcriptional level. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay further confirmed that XN could block endogenous activation of nuclear factor kappa B, a key transcription factor regulates the expression of CXCR4 in cancer cells. Consistent with the above molecular basis, XN abolished cell invasion induced by CXCL12 in both breast and colon cancer cells. Interestingly, although co-exist in hops, XN is the only isoform that exhibited the inhibitory effect on the expression of CXCR4 compared with other isomers, isoxanthohumol and 8-prenylnaringenin. Together, our results suggested that XN, as a novel inhibitor of CXCR4, could be a promising therapeutic agent contributed to cancer treatment.

The purpose of this study was to test the hypothesis that administration of epigallocatechin-3-gallate (EGCG), a polyphenol present in abundance in widely consumed tea, inhibits cell proliferation, invasion, and angiogenesis in breast cancer patients. EGCG in 400 mg capsules was orally administered three times daily to breast cancer patients undergoing treatment with radiotherapy. Parameters related to cell proliferation, invasion, and angiogenesis were analyzed while blood samples were collected at different time points to determine efficacy of the EGCG treatment. Compared to patients who received radiotherapy alone, those given radiotherapy plus EGCG for an extended time period (two to eight weeks) showed significantly lower serum levels of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and reduced activation of metalloproteinase-9 and metalloproteinase-2 (MMP9/MMP2). Addition of sera obtained from patients treated with combination of radiotherapy and EGCG feeding for 2-8 weeks to in vitro cultures of highly-metastatic human MDA-MB-231 breast cancer cells resulted in the following significant changes: (1) suppression of cell proliferation and invasion; (2) arrest of cell cycles at the G0/G1 phase; (3) reduction of activation of MMP9/MMP2, expressions of Bcl-2/Bax, c-Met receptor, NF-κB, and the phosphorylation of Akt. MDA-MB-231 cells exposed to 5-10 μM EGCG also showed significant augmentation of the apoptosis inducing effects of γ-radiation, concomitant with reduced NF-κB protein level and AKT phosphorylation. These results provide hitherto unreported evidence that EGCG potentiated efficacy of radiotherapy in breast cancer patients, and raise the possibility that this tea polyphenol has potential to be a therapeutic adjuvant against human metastatic breast cancer.

The small heat shock protein, α-crystallin, exists in two isoforms, αA and αB, and displays strong ability against stress-induced apoptosis. Regarding their functional mechanisms, we and others have demonstrated that they are able to regulate members in both caspase and Bcl-2 families. In addition, we have also shown that αA and αB may display differential anti-apoptotic mechanisms under certain stress conditions. While αA-crystallin regulates activation of the AKT signaling pathway, αB negatively regulates the MAPK pathway to suppress apoptosis induced by UV and oxidative stress. Although previous studies revealed that αA and αB could regulate members in both caspase and Bcl-2 families, the molecular mechanism, especially the in vivo regulation still waits to be elucidated. In the present communication, we present both in vitro and in vivo evidence to further demonstrate the regulation of caspase-3 and Bax by αA and αB. First, Surface Plasmon Resonance (SPR) and yeast two-hybrid selection analysis demonstrate that αA and αB directly bind to caspase-3 and Bax with differential affinities. Second, immunohistochemistry reveals that αA and αB regulate caspase-3 and Bax at different developmental stages of mouse embryo. Third, coimmunoprecipitation shows that αA and αB form in vivo interacting complexes with caspase-3 and Bax. Together, our results further confirm that αA and αB regulate caspase-3 and Bax in vitro and in vivo to regulate lens differentiation.

PIGA mutations in paroxysmal nocturnal hemoglobinuria (PNH) patients lead to a glycosylphosphatidylinositol (GPI)-linked membrane proteins expression deficiency. Herein, we report the constitutive expression of the transmembrane CD160 (CD160-TM) activating receptor on non PIGA-mutated PNH patients circulating NK cells. In healthy individuals, only the GPI-anchored isoform of CD160 receptors is expressed on the circulating NK lymphocytes, while the transmembrane isoform appears after ex vivo activation. Similarly to CD160-GPI, we identified CD160-TM as a receptor for the MHC class I molecules. We demonstrate that PNH patients NK lymphocytes spontaneously produce significant amounts of IFN-γ that is inhibited by anti-CD160-TM or anti-MHC class I mAbs. These results indicate that circulating NK cells from PNH patients exhibit a self-MHC class I molecule reactive effector function, which could be mediated through the recruitment of CD160-TM receptor. Our data provide new insights regarding the possible role of CD160-TM on PNH patients NK lymphocytes and in the pathogenesis of the disease

In patients with congenital heart defects, chromosomal anomalies are 100 times more frequent than in control subjects. Coarctation of the aorta can be detected in 15-20% of patients with Ullrich-Turner syndrome. By extensively reviewing literature involving breakpoint analysis of gonosomal deletions in Ullrich- Turner syndrome patients with and without coarctation of the aorta, we identified several gonosomal homolgous gene pairs of interest. Four of these homologous gene pairs were investigated by standard DNA sequencing in a cohort of 83 patients with non-syndromic coarctation of the aorta. Subsequently stability of mutant RNA and protein was analyzed to verify functional relevance of detected mutations. We identified two unreported missense mutations in Exon 8 (p.D69H) and 9 (p.R176W) of TBL1Y. Bioinformatic analysis and 3D modelling predicted that both mutations lead to TBL1Y loss of function. In RT-PCR and Western blot analyses of HEK293 cells transfected with a vector carrying the full-length TBL1Y (wild-type and mutant), we documented the predicted protein instability by showing protein decay for both mutant proteins. TBL1Y is similar to its gonosomal homologue, TBL1X, and its autosomal homologue, TBLR1, on chromosome 3. Both genes are part of co-repressor machineries and required for transcriptional activation by transcription factors that involve CtBP1/2, which contributes to Notch signaling. Several studies have shown that Notch signalling is important for proper development of the left ventricular outflow tract. Our findings suggest that TBL1Y is involved in the genesis of non-syndromic coarctation of the aorta.

Prevention of adverse cardiac remodeling after myocardial infarction (MI) remains a therapeutic challenge. Angiotensin-converting enzyme inhibitors (ACE-I) are a well-established first-line treatment. ACE-I delay fibrosis, but little is known about their molecular effects on cardiomyocytes. We investigated the effects of the ACE-I delapril on cardiomyocytes in a mouse model of heart failure (HF) after MI. Mice were randomly assigned to three groups: Sham, MI, and MI-D (6 weeks of treatment with a non-hypotensive dose of delapril started 24h after MI). Echocardiography and pressure-volume loops revealed that MI induced hypertrophy and dilation, and altered both contraction and relaxation of the left ventricle. At the cellular level, MI cardiomyocytes exhibited reduced contraction, slowed relaxation, increased diastolic Ca2+ levels, decreased Ca2+-transient amplitude, and diminished Ca2+ sensitivity of myofilaments. In MI-D mice, however, both mortality and cardiac remodeling were decreased when compared to non-treated MI mice. Delapril maintained cardiomyocyte contraction and relaxation, prevented diastolic Ca2+ overload and retained the normal Ca2+ sensitivity of contractile proteins. Delapril maintained SERCA2a activity through normalization of P-PLB/PLB (for both Ser16- PLB and Thr17-PLB) and PLB/SERCA2a ratios in cardiomyocytes, favoring normal reuptake of Ca2+ in the sarcoplasmic reticulum. In addition, delapril prevented defective cTnI function by normalizing the expression of PKC, enhanced in MI mice. In conclusion, early therapy with delapril after MI preserved the normal contraction/relaxation cycle of surviving cardiomyocytes with multiple direct effects on key intracellular mechanisms contributing to preserve cardiac function