Current Molecular Medicine (v.11, #6)

Editorial (A Significant Step Towards New Altitude) by David Wan-Cheng Li (437-438).
In marking its 11th anniversary, Current Molecular Medicine is undergoing significant change in its contents.Starting from this issue, CMM is going to publish research articles with exciting and important new discoveries. Inthe present issue, seven articles dealing with mechanisms of various human diseases are published.The first article was contributed by Tang et al., authors of previous articles published in PNAS, etc., whoillustrated an important development in the treatment of inflammatory diseases. Abnormal and prolongedinflammatory reaction is related to a variety of disorders such as immune system disorders, atherosclerosis,arthritis, heart disease and cancer. Previous studies have revealed the existence of a link between inflammatorydiseases and elevated levels of Tumor Necrosis Factor alpha (TNF-..). Therefore, modulation of TNF-.. expressionis important in the regulation of inflammatory disorders. In the present study, the authors demonstrated that atranscription factor named LPS-induced TNF factor (LITAF) significantly induces TNF-.. production. Furthermore,they found that p53 and its synthetic peptide 162-motif specifically downregulate LITAF/TNF-.. gene expression inhuman cells in vitro. The 162-motif, when delivered into cells and organs, reduces serum TNF-.. level in mice andprevents TNF-..-induced lung lesions and endotoxic shock. These results highlight the regulation of LITAF/TNF-.. byp53 and its short peptide 162-motif and pave the way for pharmacotherapeutic approaches in the treatment ofinflammatory diseases.The second article was contributed by Rull et al., authors of previous articles published in J. Proteome Res., etc.,who provided strong evidence for the plausible role of the combined assessment of defense against oxidative stressand inflammation in the evaluation of peripheral arterial diseases such as atherosclerosis. Generalizedatherosclerosis is likely in symptomatic peripheral arterial diseases (PAD), affecting a large portion of arteries in thelow extremities. To search for the potential indicator of disease state and/or susceptibility, the authors proposed anovel approach based on a putative role between paraoxonase-1 and monocyte chemoattractant protein-1/chemokine ligand-2. These results help to establish clinical criteria for the diagnosis of symptomatic peripheralarterial diseases.The third article was contributed by Mitchell et al., authors of previous articles published in Oncogene, etc., whoelegantly demonstrated that GEFT, a Rho family guanine nucleotide exchange factor can regulate differentiation oflens epithelial cells. This regulation occurs through a Rac1-dependent mechanism. Previously this groupdemonstrated that GEFT regulates differentiation of hippocampal neurons and neuro2a neuroblastoma cells. Herethe authors provided the first evidence to show that GEFT regulates the localization of Rac1 to modulate expressionof multiple genes of the crystallin families and thus controls differentiation of the ocular lens. These results shednew light to the functional mechanisms of GEFT in regulating cell differentiation.The 4th article was contributed by Wu group, authors of previous articles published in Proteomics, etc., whoexplored the possible molecular mechanism mediating viral infection-triggered congenital cardiovascular diseasesduring the perinatal period using microarray analysis. These authors found that 99 genes display differentialexpressions during viral infection of the ECV304 cells by Rubella virus, human Cytomegalovirus and HerpesSimplex virus. The present study suggested a possible common molecular mechanism how viral infection mightresult in congenital cardiovascular diseases.The 5th article was contributed by Xiao group, authors of previous articles published in Cancer Research, etc.,who explored the role of adipocytes in regulating osteoblast differentiation during bone aging. Using direct andindirect culture modes, the authors revealed presence of the crosstalk between adipocytes and osteoblasts throughsecretory factors in the medium. After comparative analysis of gene expression in the conditioned osteoblasts andadipocytes, the authors suggested that the adipocytes derived from bone mesenchymal stem cells may regulateosteoblast differentiation in the aged bone through TGF-....mediated cranonical Wnt signaling pathway.The 6th article was contributed by Wu group, authors of previous articles published in Plos One, etc., whoexplored the role of docosahexaenoic acid (DHA) in sensitizing brain tumor cells to etoposide-induced apoptosis.These authors demonstrated that when combined, DHA and etopside markedly suppress expression of the genesinvolved in DNA damage repair, cell proliferation, survival, invasion, and angiogenesis and suggested that it may bebeneficial for combined therapy on brain tumors with the anticancer drugs DHA and etoposide.The last article was contributed by Jankun and Skrzypczak-Jankun, authors of previous articles published inCancer Research, etc., who reported the single nucleotide polymorphisms in the plasminogen activator inhibitor(PAI-1). These authors thoroughly discussed the functional importance of different mutations in PAI-1. These resultsare important for us to have a full understanding of the blood clotting process.These articles, out of questions, will contribute to important aspects of the novel mechanisms for various humandiseases as well as important information for the development of new therapeutic strategies for these diseases.

p53 Peptide Prevents LITAF-Induced TNF-Alpha-Mediated Mouse Lung Lesions and Endotoxic Shock by X. Tang, A. O'Reilly, M. Asano, J.C. Merrill, K.K. Yokoyama, S. Amar (439-452).
Abnormal and prolonged inflammatory reaction is seen in a wide variety of disorders, and high levelof Tumor Necrosis Factor alpha (TNF-α) has been linked to these disorders. Therefore, modulation of TNF-αexpression is important in the regulation of inflammatory disorders. In our previous study, we have shown thata transcription factor LPS-induced TNF factor (LITAF) significantly induces TNF-α production. Furthermore, wefound that p53 and its synthetic peptide 162-motif specifically downregulate LITAF/TNF-α gene expression inhuman cells in vitro. Thus, in the present study, the role of p53 in regulating TNF-α-mediated inflammation wasinvestigated. Our data showed that a synthetic peptide, named 162-motif, corresponding to this regionfunctions independently from p53 to cause a significant suppression of TNF-α gene expression in mouseprimary macrophages. The 162-motif, when delivered into cells and organs, reduces serum TNF-α level inmice and prevents TNF-α-induced lung lesions and endotoxic shock. Our findings highlight the regulation ofLITAF/TNF-α by p53 and its short peptide 162-motif. These in vitro and in vivo observations serve to pave theway for pharmacotherapeutic approaches in the treatment of inflammatory diseases.

The Role of Combined Assessment of Defense Against Oxidative Stress and Inflammation in the Evaluation of Peripheral Arterial Disease by A. Rull, R. Garcia, L. Fernandez-Sender, R. Beltran-Debon, G. Aragones, J.M. Alegret, C. Alonso-Villaverde, B. Mackness, M. Mackness, J.Camps, V. Martin-Paredero, J. Joven (453-464).
Atherosclerosis in symptomatic peripheral arterial disease affects wide portions of numerous arteriesin lower extremities. The resulting active inflammation in a considerable amount of arterial tissue facilitatessystemic detection via measurement of inflammation-related variables. We reasoned that the combinedassessment of defense against oxidative stress, in the form of paraoxonase-1 (PON1), and monocytemigration measured as circulating (C-C motif) ligand 2 (CCL2), may play a role in the evaluation of thesepatients. Plasma CCL2 and serum PON1-related variables, assessed by their interaction with functionalgenetic variants, were measured in a cross-sectional study in patients with symptomatic PAD. We found thatPON1 activity and concentration were significantly lower and CCL2 concentration higher in PAD patientscompared to controls, that the combination of plasma CCL2 and PON1- related values, especially PON1concentration differentiated, almost perfectly, controls from patients and that the expression of CCL2 andPON1 generally co-localized in the atherosclerotic lesion. Since no association with genetic variants wasfound, such a relationship is probably the result of the disease. Our data suggest a coordinated role betweenCCL2 and PON1 that may be detected in blood with simple measurements and may represent an indicator ofthe extent of atherosclerosis.

GEFT, A Rho Family Guanine Nucleotide Exchange Factor, Regulates Lens Differentiation through a Rac1-Mediated Mechanism by D.C. Mitchell, B.A. Bryan, L. Liu, X.-H. Hu, X.-Q. Huang, W.-K. Ji, P.-C. Chen, W.-F. Hu, J-P Liu, J. Zhang, M. Liu, D.W.-C. Li (465-480).
The Rho-family of small GTPase specific guanine nucleotide exchange factor, GEFT, is expressedat high levels in adult human excitable tissues including the brain, heart, and skeletal muscle. Previously, wedemonstrated that GEFT is specifically expressed in the adult mouse hippocampus and cerebellum, and thatoverexpression of this protein can result in neurite and dendrite remodeling. This finding prompted us toexplore the expression of GEFT in other tissues, which share common developmental ancestry to the nervoussystem, specifically the ocular system. Using immunohistochemical analysis specific for GEFT proteinexpression, we observed the highest ocular expression of GEFT occurring in the neuroblastic layer anddifferentiating lens fibers of the late-stage mouse embryo, and in the postnatal corneal epithelium, lensepithelium, and throughout the retina. Exogenous expression of GEFT in N/N1003A rabbit lens epithelial cellsinduced lens fiber differentiation as reflected by cell elongation and lentoid formation, as well as a strongincrease in β-crystallin and filensin expression. Moreover, transfection of lens epithelial cells with GEFTresulted in a Rac-1 mediated up-regulation of αA-, αB-, βB-, γC-, or γF-crystallin promoter activities that is inpart dependent on the nuclear localization of Rac1. Furthermore, pharmacological inhibition of Rac1 blockedGEFT-induced N/N1003A lens fiber differentiation and βB-crystallin expression in ex vivo mouse lens explants.These results demonstrate for the first time a role for GEFT in lens cell differentiation and mouse eyedevelopment. Moreover, GEFT regulation of lens differentiation and eye development occurs through a Rac1-dependent mechanism.

Microarray Profiling Analysis Uncovers Common Molecular Mechanisms of Rubella Virus, Human Cytomegalovirus, and Herpes Simplex Virus Type 2 Infections in ECV304 Cells by X. Mo, L. Xu, Q. Yang, H. Feng, J. Peng, Y. Zhang, W. Yuan, Y. Wang, Y. Li, Y. Deng, Y. Wan, Z. Chen, F. Li, X. Wu (481-488).
To study the common molecular mechanisms of various viruses infections that might result incongential cardiovascular diseases in perinatal period, changes in mRNA expression levels of ECV304 cellsinfected by rubella virus (RUBV), human cytomegalovirus (HCMV), and herpes simplex virus type 2 (HSV-2)were analyzed using a microarray system representing 18,716 human genes. 99 genes were found to exhibitdifferential expression (80 up-regulated and 19 down-regulated). Biological process analysis showed that 33signaling pathways including 22 genes were relevant significantly to RV, HCMV and HSV-II infections. Of these33 biological processes, 28 belong to one-gene biological processes and 5 belong to multiple-gene biologicalprocesses. Gene annotation indicated that the 5 multiple-gene biological processes including regulation of cellgrowth, collagen fibril organization, mRNA transport, cell adhesion and regulation of cell shape, and sevendown- or up-regulated genes [CRIM1 (cysteine rich transmembrane BMP regulator 1), WISP2 (WNT1 induciblesignaling pathway protein 2), COL12A1 (collagen, type XII, alpha 1), COL11A2 (collagen, type XI, alpha 2),CNTN5 (contactin 5), DDR1 (discoidin domain receptor tyrosine kinase 1), VEGF (vascular endothelial growthfactor precursor)], are significantly correlated to RUBV, HCMV and HSV-2 infections in ECV304 cells. Theresults obtained in this study suggested the common molecular mechanisms of viruses infections that mightresult in congential cardiovascular diseases.

Adipocytes Derived from Human Bone Marrow Mesenchymal Stem Cells Exert Inhibitory Effects on Osteoblastogenesis by H. Zhang, W. Lu, Y. Zhao, P. Rong, R. Cao, W. Gu, J. Xiao, D. Miao, J. Lappe, R. Recker, G.G. Xiao (489-502).
The infiltration of adipocytes in osteoporotic patients' bone marrow suggests an important regulatoryfunction of bone marrow fat on the development of aged bone. Therefore, we have examined the effects ofadipocytes derived from bone mesenchymal stem cell (MSC) on osteoblast differentiation using two differentco-culture modes (direct mode and indirect mode). Alkaline phosphatase (ALP)-positive areas and mineralizedareas of MSC-derived osteoblasts decrease similarly in the two co-culture modes as the amount of MSCderivedadipocytes increases, suggesting that the crosstalk between adipocytes and osteoblasts may bemainly through secretory factors in the medium. To further understand the molecular mechanisms, both mRNAand protein expressions in osteoblasts in the lower layer of the indirect mode were analyzed, leading toidentification of 12 differential genes/proteins. Among them, S100A6 and calreticulin are possibly related tobone formation. S100A6 was down-regulated and calreticulin was up-regulated as MSC-derived adipocytesincreased. Similarly, differential expression of these proteins was also observed in bone tissue slides fromyoung (1-month-old) and old (6-month-old) mice. The expression level of β-catenin in osteoblasts of bonetissues was lower in 6-month-old mice compared to 1-month-old mice. Total TGF-β analyzed with antibodybasedprotein microarray and active TGF-β analyzed with ELISA in the co-cultured cell medium increasedconsistently as the amount of adipocytes increased. Taken together, our results suggest that MSC-derivedadipocytes may regulate osteoblast differentiation in the aged bone through TGF-β-mediated canonical Wntsignaling.

Docosahexaenoic Acid (DHA) Sensitizes Brain Tumor Cells to Etoposide-Induced Apoptosis by F. Wang, K. Bhat, M. Doucette, S. Zhou, Y. Gu, B. Law, X. Liu, E.T. Wong, J.X. Kang, T.-C. Hsieh, S.Y. Qian, E. Wu (503-511).
In this study, we investigated whether DHA, a nutritionally important n-3 unsaturated fatty acid,modulated the sensitivity of brain tumor cells to the anticancer drug, etoposide (VP16). Medulloblastoma (MB)cell lines, Daoy and D283, and glioblastoma (GBM) cell lines, U138 and U87, were exposed to DHA or VP16alone or in combination. The effects on cell proliferation and the induction of apoptosis were determined byusing MTS and Hoechest 33342/PI double staining. U87 and U138 cells were found to be insensitive to theaddition of DHA and VP16, whereas the two MB cell lines showed high sensitivity. DHA or VP16 alone showedlittle effect on cell proliferation or death in either the MB or GBM cell lines, but pretreatment with DHAenhanced the responsiveness to VP16 in the MB cell lines. To understand the mechanisms of combined DHAand VP16 on MB cells, pathway specific oligo array analyses were performed to dissect possible signalingpathways involved. The addition of DHA and VP16, in comparison to VP16 added alone, resulted in markedsuppression in the expression of several genes involved in DNA damage repair, cell proliferation, survival,invasion, and angiogenesis, including PRKDC, Survivin, PIK3R1, MAPK14, NFκB1, NFκBIA, BCL2, CD44, andMAT1. These results suggest (1) that the effects of DHA and VP16 in brain tumor cells are mediated in part bythe down regulation of events involved in DNA repair and the PI3K/MAPK signaling pathways and (2) that braintumors genotypically mimicked by MB cells may benefit from therapies combining DHA with VP16.

Plasminogen activator inhibitor (PAI-1) is a fast acting inhibitor of tissue and urokinase plasminogenactivators (tPA and uPA). In that way PAI-1 regulates proteolytic activity of many physiological and pathologicalprocesses [1-3]. PAI-1 plays an important role in blood coagulation controlling clot lysis which is triggered bytPA activated plasminogen [4]. Only two types of mutations are reported to be associated with PAI-1; one is theframe-shift mutation in exon 4 of PAI-1 gene resulting in a truncated nonfunctional protein and in complete PAI-1 deficiency. The other SNP causes Ala15Thr mutation in the signal peptide. A literature search revealed fivevariants of polymorphisms during a study of over one thousand individuals. Two are associated withthrombophilia (765 4G/5G and -844 A>G, in the promoter), risk of myocardial infarction and postoperative deepvenous thrombosis related to higher than normal levels of PAI-1. The other SNPs associated with PAI-1deficiency are Ala15Thr, Val17Ile and they are located in the central hydrophobic core of the PAI-1 signalpeptide of PAI-1 and Asn195Ile in the ‘A’ - sheet of the PAI-1. We have analyzed two SNPs not reported to beassociated with PAI-1 deficiency. Our analysis suggests that Val17Ile PAI-1 variant might cause slower PAI-1secretion leading to the deficiency at time and place where it is needed in a similar way as for Ala15Thr SNP.The Asn195Ile mutant may be more stable only as latent form thus no PAI-1 deficiency is expected in thismutant.