IL-17 stimulates MMP-1 expression in primary human cardiac fibroblasts via p38 MAPK- and ERK1/2-dependent C/EBP-β, NF-κB, and AP-1 activation

2007 ◽  
Vol 293 (6) ◽  
pp. H3356-H3365 ◽  
Author(s):  
Dolores M. Cortez ◽  
Marc D. Feldman ◽  
Srinivas Mummidi ◽  
Anthony J. Valente ◽  
Bjorn Steffensen ◽  
...  
Keyword(s):  
Transcription Factor ◽  
P38 Mapk ◽  
Western Blotting ◽  
Cardiac Fibroblasts ◽  
Critical Role ◽  
Dominant Negative ◽  
Expression Vectors ◽  
Type I ◽  
Mrna Levels ◽  
Human Cardiac Fibroblasts

Matrix metalloproteinases (MMPs) degrade collagen and mediate tissue remodeling. The novel cytokine IL-17 is expressed during various inflammatory conditions and modulates MMP expression. We investigated the effect of IL-17 on MMP-1 expression in primary human cardiac fibroblasts (HCF) and delineated the signaling pathways involved. HCF were treated with recombinant human IL-17. MMP-1 expression was analyzed by Northern blotting, RT-quantitative PCR, Western blotting, and ELISA; transcriptional induction and transcription factor binding by EMSA, ELISA, and reporter assay; and p38 MAPK and ERK1/2 activation by protein kinase assays and Western blotting. Signal transduction pathways were investigated using pharmacological inhibitors, small interfering RNA (siRNA), and adenoviral dominant-negative expression vectors. IL-17 stimulated MMP-1 gene transcription, net mRNA levels, protein, and promoter-reporter activity in HCF. This response was blocked by IL-17 receptor-Fc chimera and IL-17 receptor antibodies, but not by IL-6, TNF-α, or IL-1β antibodies. IL-17-stimulated type I collagenase activity was inhibited by the MMP inhibitor GM-6001 and by siRNA-mediated MMP-1 knockdown. IL-17 stimulated activator protein-1 [AP-1 (c-Fos, c-Jun, and Fra-1)], NF-κB (p50 and p65), and CCAAT enhancer-binding protein (C/EBP)-β DNA binding and reporter gene activities, effects attenuated by antisense oligonucleotides, siRNA-mediated knockdown, or expression of dominant-negative signaling proteins. Inhibition of AP-1, NF-κB, or C/EBP activation attenuated IL-17-stimulated MMP-1 expression. IL-17 induced p38 MAPK and ERK1/2 activation, and inhibition by SB-203580 and PD-98059 blunted IL-17-mediated transcription factor activation and MMP-1 expression. Our data indicate that IL-17 induces MMP-1 in human cardiac fibroblasts directly via p38 MAPK- and ERK-dependent AP-1, NF-κB, and C/EBP-β activation and suggest that IL-17 may play a critical role in myocardial remodeling.

Abstract 1058: Novel Regulation of Wnt-Inducible Secreted Protein 1 by TNF-alpha in Primary Human Cardiac Fibroblasts

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
James T Colston ◽  
Sam D de la Rosa ◽  
Steven R Bailey ◽  
Bysani Chandrasekar
Keyword(s):  
Dna Binding ◽  
Western Blotting ◽  
Cardiac Fibroblasts ◽  
Critical Role ◽  
Secreted Protein ◽  
Dependent Manner ◽  
Potent Inducer ◽  
Reporter Activity ◽  
Creb Activation ◽  
Human Cardiac Fibroblasts

Wnt1-induced secreted protein-1 (WISP-1) is a member of the CCN family of growth factors known to play a role in cellular growth, transformation, and survival. We previously demonstrated that WISP-1 is upregulated in the heart post-infarct, stimulates fibroblast proliferation and collagen expression, and is induced by the proinflammatory cytokine TNF-a. The present study was designed to investigate the molecular mechanisms involved in TNF-a-mediated WISP-1 induction in primary human cardiac fibroblasts (hCF). hCF were treated with rhTNF-a. WISP-1 expression was analyzed by Northern, Western and promoter-reporter assays. ERK1/2 and JNK activations by Western blotting using activation-specific antibodies and imunecomplex kinase assays. CREB activation by Western blotting, EMSA and reporter assay. Results demonstrate that TNF-a potently induces WISP-1 mRNA and protein expression in a time- and dose-dependent manner, effects that were blocked by anti-TNFR2, but not anti-TNFR1, neutralizing antibodies. Further investigations revealed that TNF-a stimulates WISP-1 promoter-reporter activity, an effect that was blunted when the core CREB DNA binding sequence was mutated or following the overexpression of dnCREB. TNF-a treatment induced CREB phosphorylation, in vitro and in vivo DNA binding, and reporter gene activities. TNF-a also induced ERK1/2 phosphorylation and activity, effects that were blocked by the MEK1 inhibitor U0126 and the ERK1/2 inhibitor PD98059. Furthermore, inhibition of ERK1/2 blunted CREB activation while inhibition of ERK1/2 and overexpression of dnCREB attenuated TNF-a-mediated WISP-1 promoter-reporter activity and mRNA expression. However, inhibition of JNK by SP600125 failed to modulate TNF-a-mediated WISP-1 induction. Most importantly, siRNA-mediated WISP-1 knockdown attenuated TNF-a-mediated hCF proliferation. Thus, our results demonstrate for the first time that TNF-a is a potent inducer of WISP1 expression in hCF, and TNF-a induces WISP-1 expression via TNFR2, MEK1, ERK, and CREB. Since TNF-a and WISP-1 are upregulated post-infarct, our results indicate that TNF-a/WISP-1 signaling may play a critical role in post-infarct myocardial remodeling.

scholarly journals The AP-1 Transcription Factor Fosl-2 Regulates Autophagy in Cardiac Fibroblasts during Myocardial Fibrogenesis

2021 ◽  
Vol 22 (4) ◽  
pp. 1861
Author(s):  
Jemima Seidenberg ◽  
Mara Stellato ◽  
Amela Hukara ◽  
Burkhard Ludewig ◽  
Karin Klingel ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Molecular Mechanisms ◽  
Cardiac Fibrosis ◽  
Type I Collagen ◽  
Transforming Growth Factor ◽  
Cardiac Fibroblasts ◽  
Smooth Muscle Actin ◽  
Beclin 1 ◽  
Type I ◽  
Alpha Smooth Muscle Actin

Background: Pathological activation of cardiac fibroblasts is a key step in development and progression of cardiac fibrosis and heart failure. This process has been associated with enhanced autophagocytosis, but molecular mechanisms remain largely unknown. Methods and Results: Immunohistochemical analysis of endomyocardial biopsies showed increased activation of autophagy in fibrotic hearts of patients with inflammatory cardiomyopathy. In vitro experiments using mouse and human cardiac fibroblasts confirmed that blockade of autophagy with Bafilomycin A1 inhibited fibroblast-to-myofibroblast transition induced by transforming growth factor (TGF)-β. Next, we observed that cardiac fibroblasts obtained from mice overexpressing transcription factor Fos-related antigen 2 (Fosl-2tg) expressed elevated protein levels of autophagy markers: the lipid modified form of microtubule-associated protein 1A/1B-light chain 3B (LC3BII), Beclin-1 and autophagy related 5 (Atg5). In complementary experiments, silencing of Fosl-2 with antisense GapmeR oligonucleotides suppressed production of type I collagen, myofibroblast marker alpha smooth muscle actin and autophagy marker Beclin-1 in cardiac fibroblasts. On the other hand, silencing of either LC3B or Beclin-1 reduced Fosl-2 levels in TGF-β-activated, but not in unstimulated cells. Using a cardiac hypertrophy model induced by continuous infusion of angiotensin II with osmotic minipumps, we confirmed that mice lacking either Fosl-2 (Ccl19CreFosl2flox/flox) or Atg5 (Ccl19CreAtg5flox/flox) in stromal cells were protected from cardiac fibrosis. Conclusion: Our findings demonstrate that Fosl-2 regulates autophagocytosis and the TGF-β-Fosl-2-autophagy axis controls differentiation of cardiac fibroblasts. These data provide a new insight for the development of pharmaceutical targets in cardiac fibrosis.

scholarly journals BMP15 Suppresses Progesterone Production by Down-Regulating StAR via ALK3 in Human Granulosa Cells

2013 ◽  
Vol 27 (12) ◽  
pp. 2093-2104 ◽  
Author(s):  
Hsun-Ming Chang ◽  
Jung-Chien Cheng ◽  
Christian Klausen ◽  
Peter C. K. Leung
Keyword(s):  
Granulosa Cells ◽  
Critical Role ◽  
Regulatory Protein ◽  
Granulosa Cell Tumor ◽  
Type I ◽  
Mrna Levels ◽  
Protein Levels ◽  
Human Granulosa Cells ◽  
Progesterone Production ◽  
Steroidogenic Acute Regulatory

In addition to somatic cell-derived growth factors, oocyte-derived growth differentiation factor (GDF)9 and bone morphogenetic protein (BMP)15 play essential roles in female fertility. However, few studies have investigated their effects on human ovarian steroidogenesis, and fewer still have examined their differential effects or underlying molecular determinants. In the present study, we used immortalized human granulosa cells (SVOG) and human granulosa cell tumor cells (KGN) to compare the effects of GDF9 and BMP15 on steroidogenic enzyme expression and investigate potential mechanisms of action. In SVOG cells, neither GDF9 nor BMP15 affects the mRNA levels of P450 side-chain cleavage enzyme or 3β-hydroxysteroid dehydrogenase. However, treatment with BMP15, but not GDF9, significantly decreases steroidogenic acute regulatory protein (StAR) mRNA and protein levels as well as progesterone production. These suppressive effects, along with the induction of Sma and Mad-related protein (SMAD)1/5/8 phosphorylation, are attenuated by cotreatment with 2 different BMP type I receptor inhibitors (dorsomorphin and DMH-1). Furthermore, depletion of activin receptor-like kinase (ALK)3 using small interfering RNA reverses the effects of BMP15 on SMAD1/5/8 phosphorylation and StAR expression. Similarly, knockdown of ALK3 abolishes BMP15-induced SMAD1/5/8 phosphorylation in KGN cells. These results provide evidence that oocyte-derived BMP15 down-regulates StAR expression and decreases progesterone production in human granulosa cells, likely via ALK3-mediated SMAD1/5/8 signaling. Our findings suggest that oocyte may play a critical role in the regulation of progesterone to prevent premature luteinization during the late stage of follicle development.

scholarly journals Keratin 18 is an integral part of the intermediate filament network in murine skeletal muscle

2020 ◽  
Vol 318 (1) ◽  
pp. C215-C224 ◽  
Author(s):  
Joaquin M. Muriel ◽  
Andrea O’Neill ◽  
Jaclyn P. Kerr ◽  
Emily Kleinhans-Welte ◽  
Richard M. Lovering ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Isometric Force ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Type I ◽  
Mrna Levels ◽  
Force Transmission ◽  
Keratin 18 ◽  
Murine Skeletal Muscle

Intermediate filaments (IFs) contribute to force transmission, cellular integrity, and signaling in skeletal muscle. We previously identified keratin 19 (Krt19) as a muscle IF protein. We now report the presence of a second type I muscle keratin, Krt18. Krt18 mRNA levels are about half those for Krt19 and only 1:1,000th those for desmin; the protein was nevertheless detectable in immunoblots. Muscle function, measured by maximal isometric force in vivo, was moderately compromised in Krt18-knockout ( Krt18-KO) or dominant-negative mutant mice ( Krt18 DN), but structure was unaltered. Exogenous Krt18, introduced by electroporation, was localized in a reticulum around the contractile apparatus in wild-type muscle and to a lesser extent in muscle lacking Krt19 or desmin or both proteins. Exogenous Krt19, which was either reticular or aggregated in controls, became reticular more frequently in Krt19-null than in Krt18-null, desmin-null, or double-null muscles. Desmin was assembled into the reticulum normally in all genotypes. Notably, all three IF proteins appeared in overlapping reticular structures. We assessed the effect of Krt18 on susceptibility to injury in vivo by electroporating siRNA into tibialis anterior (TA) muscles of control and Krt19-KO mice and testing 2 wk later. Results showed a 33% strength deficit (reduction in maximal torque after injury) compared with siRNA-treated controls. Conversely, electroporation of siRNA to Krt19 into Krt18-null TA yielded a strength deficit of 18% after injury compared with controls. Our results suggest that Krt18 plays a complementary role to Krt19 in skeletal muscle in both assembling keratin-based filaments and transducing contractile force.

scholarly journals Effect of ALDH2 on High Glucose-Induced Cardiac Fibroblast Oxidative Stress, Apoptosis, and Fibrosis

2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Xiaoyu Gu ◽  
Tingting Fang ◽  
Pinfang Kang ◽  
Junfeng Hu ◽  
Ying Yu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Protein Expression ◽  
High Glucose ◽  
Collagen Type ◽  
Cardiac Fibroblasts ◽  
Collagen Type I ◽  
Cardiac Fibroblast ◽  
Type I ◽  
Mrna Levels ◽  
Neonate Rat

Our study aimed firstly to observe whether ALDH2 was expressed in neonate rat cardiac fibroblasts, then to investigate the effect of activation of ALDH2 on oxidative stress, apoptosis, and fibrosis when cardiac fibroblasts were subjected to high glucose intervention. Cultured cardiac fibroblasts were randomly divided into normal (NG), NG + Alda-1, high glucose (HG), HG + Alda-1, HG + Alda-1 + daidzin, HG + daidzin, and hypertonic groups. Double-label immunofluorescence staining, RT-PCR, and Western blot revealed ALDH2 was expressed in cardiac fibroblasts. Compared with NG, ALDH2 activity and protein expression were reduced, and cardiac fibroblast proliferation, ROS releasing, 4-HNE protein expression, collagen type I and III at mRNA levels, and the apoptosis rate were increased in HG group. While in HG + Alda-1 group, with the increases of ALDH2 activity and protein expression, the cardiac fibroblast proliferation and ROS releasing were decreased, and 4-HNE protein expression, collagen type I and III at mRNA levels, and apoptosis rate were reduced compared with HG group. When treated with daidzin in HG + Alda-1 group, the protective effects were inhibited. Our findings suggested that ALDH2 is expressed in neonate rat cardiac fibroblasts; activation of ALDH2 decreases the HG-induced apoptosis and fibrosis through inhibition of oxidative stress.

scholarly journals Helicobacter pylori VacA Enhances Prostaglandin E2 Production through Induction of Cyclooxygenase 2 Expression via a p38 Mitogen-Activated Protein Kinase/Activating Transcription Factor 2 Cascade in AZ-521 Cells

2007 ◽  
Vol 75 (9) ◽  
pp. 4472-4481 ◽  
Author(s):  
Junzo Hisatsune ◽  
Eiki Yamasaki ◽  
Masaaki Nakayama ◽  
Daisuke Shirasaka ◽  
Hisao Kurazono ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Transcription Factor ◽  
Protein Kinase ◽  
P38 Mapk ◽  
Cyclooxygenase 2 ◽  
Mitogen Activated Protein Kinase ◽  
Mrna Levels ◽  
Mitogen Activated Protein ◽  
Cox 2 ◽  
Activating Transcription Factor

ABSTRACT Treatment of AZ-521 cells with Helicobacter pylori VacA increased cyclooxygenase 2 (COX-2) mRNA in a time- and dose-dependent manner. A p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, blocked elevation of COX-2 mRNA levels, whereas PD98059, which blocks the Erk1/2 cascade, partially suppressed the increase. Consistent with involvement of p38 MAPK, VacA-induced accumulation of COX-2 mRNA was reduced in AZ-521 cells overexpressing a dominant-negative p38 MAPK (DN-p38). Phosphatidylinositol-specific phospholipase C, which inhibits VacA-induced p38 MAPK activation, blocked VacA-induced COX-2 expression. In parallel with COX-2 expression, VacA increased prostaglandin E2 (PGE2) production, which was inhibited by SB203580 and NS-398, a COX-2 inhibitor. VacA-induced PGE2 production was markedly attenuated in AZ-521 cells stably expressing DN-p38. VacA increased transcription of a COX-2 promoter reporter gene and activated a COX-2 promoter containing mutated NF-κB or NF-interleukin-6 sites but not a mutated cis-acting replication element (CRE) site, suggesting direct involvement of the activating transcription factor 2 (ATF-2)/CREB-binding region in VacA-induced COX-2 promoter activation. The reduction of ATF-2 expression in AZ-521 cells transformed with ATF-2-small interfering RNA duplexes resulted in suppression of COX-2 expression. Thus, VacA enhances PGE2 production by AZ-521 cells through induction of COX-2 expression via the p38 MAPK/ATF-2 cascade, leading to activation of the CRE site in the COX-2 promoter.

Enforced STAT3 and PU.1 Expression Activates Type I Interferon-Producing Cell and Dendritic Cell Differentiation Programs in Megakaryocyte/Erythrocyte Progenitors.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 201-201
Author(s):  
Nobuyuki Onai ◽  
Aya Onai ◽  
Roxane Tussiwand ◽  
Antonio Lanzavecchia ◽  
Markus G Manz
Keyword(s):  
Transcription Factors ◽  
Dendritic Cell ◽  
Expression Vectors ◽  
Cytokine Signaling ◽  
Type I ◽  
Mrna Levels ◽  
Developmental Potential ◽  
Cell Fate Decisions ◽  
Dendritic Cell Differentiation ◽  
Flt3 Expression

Abstract A standing question in early hematopoiesis is whether cytokine signaling is sufficient to induce cell fate decisions. Previously, we have reported that enforced expression of human Flt3 in Flt3 negative megakaryocyte/erythrocyte progenitors (MEPs) rescues their interferon producing cell (IPC) and dendritic cell (DC) developmental potential: Human Flt3-signaling in MEPs leads to up-regulation of IPC, DC, and myelomonocytic development affiliated genes such as, STAT3, PU.1, and G-CSFR/M-CSFR/GM-CSFR, and activates differentiation to these lineages. To test whether single Flt3 downstream activated genes would be sufficient to cause the same effects, we transduced murine STAT3 and PU.1 cDNA into MEPs using retrovirus expression vectors, respectively. Both STAT3- and PU.1-transduced MEPs were capable to differentiate into IPCs and DCs in SCF and TPO supplemented media, and, with even higher efficiency, in SCF, TPO, and Flt3L supplemented cultures. While human Flt3-transduced MEPs maintained megakaryocyte-erythrocyte developmental potential and gained IPC, DC, and myelomonocytic cell potential, STAT3- and PU.1-transduced MEPs lost both megakaryocyte and erythrocyte developmental options, indicating that strong signaling of these transcription factors fully converted MEPs to the IPC, DC, and myelomonocytic lineages. Consistently, GATA-1 expression was down-regulated, and C/EBPα mRNA was up-regulated in STAT3- and PU.1-transduced MEPs. Interestingly, STAT3- and PU.1 over-expression in MEPs led to up-regulation of Flt3 mRNA levels, suggesting a self-sustaining effect of Flt3 signaling-cascade induced Flt3 expression. Thus, enforced expression of STAT3 and PU.1 in MEPs reprogrammed them to differentiate into IPCs, DCs and myelomonocytic cell lineages and inhibit Meg/E-lineage potential. Based on these data, we propose a model where Flt3 positive progenitor cells that locate in Flt3L rich environments will be directed to develop into IPCs and DCs. This process might be enhanced by a self-sustaining mechanism where Flt3 downstream transcription factors as STAT3 and PU.1 in turn maintain Flt3 expression.

TPO-Induced miR-150 Down-Regulates c-Myb in Primary Megakaryocytes and a Megakaryocytic Cell Line.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1238-1238 ◽  
Author(s):  
Charlene F. Barroga ◽  
Hang Pham ◽  
Kenneth Kaushansky
Keyword(s):  
Transcription Factor ◽  
Western Blotting ◽  
Luciferase Activity ◽  
Mrna Translation ◽  
Negative Control ◽  
Luciferase Reporter ◽  
Translation Efficiency ◽  
Mrna Levels ◽  
Protein Levels ◽  
Micro Rnas

Abstract Mice harboring c-Myb hypomorphic mutations display enhanced thrombopoiesis because of increased numbers of megakaryocytic progenitors (CFU-MK) and mature megakaryocytes (MK). Thrombopoietin (Tpo), the primary regulator of megakaryopoiesis, induces these same effects, which lead us to hypothesize that Tpo might act, at least in part, through modulation of c-Myb expression. We found using quantitative (Q)-PCR that c-Myb mRNA levels were 13-fold reduced during Tpo-induced MK maturation. Micro RNAs (miRs) are ∼22 nucleotide species that down-regulate gene expression by binding to the 3′ untranslated region (UTR) of specific mRNAs, enhancing mRNA degradation, or by reducing mRNA translation efficiency. We noted that the 3′UTR of c-Myb contains a number of miR target sites, including four that bind miR150; using a specific Q-PCR assay we also found that Tpo increased mir-150 expression to 160% of baseline at 24 hr and 250% at 48 hr in UT7/TPO cells (n=2 experiments). To test if miR150 affects c-Myb expression, we introduced the 3′UTR of c-Myb into a luciferase reporter gene (pCMV-luc-3′UTRcMyb), in which CMV promoter-driven luciferase activity would reflect the stability of the 3′UTR of c-Myb, and allow us to test the effects of miR150 on c-Myb expression in transduced cells; Q-PCR and western blotting were used to simultaneously assess endogenous c-Myb mRNA and protein levels in the cells treated with miR-150 and anti-miR-150, and their respective controls (Ambion, ABI). Co-transfection of UT7/TPO cells with pCMV-luc-3′UTRcMyb and miR-150 significantly down-regulated luciferase activity to 40% of baseline 24 hr following transfection (p = 0.035; n=2 experiments) compared to a miR negative control. Luciferase activity in cells transfected with a control luc plasmid lacking the 3′UTR of c-Myb was not modulated by introduction of miR-150. Q-PCR analysis revealed that endogenous c-Myb mRNA was significantly down-regulated to 60% of baseline upon transfection of miR-150 compared to the negative control (p = 0.043), while the essential megakaryocytic transcription factor, AML1/RUNX1, remained unaltered. Western blotting of these cell lysates revealed that c-Myb protein expression was down-regulated to 30% of baseline (n=3 experiments) following transduction with miR150 but not with the miR negative control. Converse experiments utilizing anti-miRs, which inhibit expression of endogenous miRs, revealed that anti-miR150 significantly upregulated luciferase activity to 180% of baseline compared to an anti-miR-negative control (p=0.003; n=2 experiments). These findings establish that miR-150 down-modulates c-Myb mRNA, and to a greater extent protein levels, suggesting effects on both mRNA stability and protein translation efficiency. And since Tpo affects miR-150 expression, our results also suggest that in addition to direct effects on the survival and growth of MK progenitor cells, mediated by the JAK/STAT, PI3K/Akt and MAPK pathways, Tpo down-modulates c-Myb expression during megakaryopoiesis through the induction of miR150. We are currently ascertaining the in vivo role of miR-150 in Tpo-induced megakaryopoiesis, but these studies already establish that hematopoietic growth factors such as Tpo can influence transcription factor expression through modulation of microRNA species.

Mir-23b Plays a Critical Role As a Tumor Suppressor miRNA In Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 122-122 ◽  
Author(s):  
Mariateresa Fulciniti ◽  
Nicola Amodio ◽  
Rajya Bandi ◽  
Rao H. Prabhala ◽  
Sophia Adamia ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Transcription Factor ◽  
Tumor Suppressor ◽  
Critical Role ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Myeloma Cells ◽  
Equity Ownership

Abstract Deregulated expression of microRNAs (miR) is a hallmark of cancer. Tumor suppressor miRNAs are generally down-regulated in cancer cells compared to their normal counterpart, and their enforced expression indeed represents a promising strategy for cancer treatment. We have found miR-23b to be downregulated in CD138+ myeloma cells from 38 multiple myeloma (MM) patients and 18 plasma cell leukemia (PCL) patients compared to normal PCs. Decreased expression of miR-23b was further confirmed in an independent dataset of 66 MM patients by TaqMan miRNA assays. The downregulation of miR-23b expression was also observed in several myeloma cells lines when compared with PBMC and BMSC. Interestingly, interaction of BMSC with MM cells resulted in further decrease in miR-23b expression in both cell types. Moreover, Interleukin-6 (IL-6) also suppressed the expression of miR-23b in a time- and dose- dependent pattern, indicating that the human bone marrow microenvironment (huBMM) modulates miR-23b levels. miR-23b is commonly repressed in autoimmune conditions by IL-17, a cytokine shown to promote myeloma cell growth and inhibit its immune function. We have indeed observed further decrease in miR-23b expression in MM cells after IL-17 treatment for 24 hours. We have also observed downregulation of miR-23b in CD19+ Waldenstrom’s Macroglobulinemia (WM) cells compared to CD19+ B cells from healthy donors, which was further decreased in the presence of components of the WM bone marrow milieu. We further assessed the functional significance of miR-23b by both gain- and loss-of-function studies. A significant decrease in cell proliferation and survival, along with induction of caspase 3/7 activity was observed over time in miR-23b mimic–transfected myeloma (H929, KMS11) and WM cell lines (MWCL1) with low miR-23b expression. At the molecular level, we have identified Sp1, a transcription factor endowed with oncogenic activity in MM and WM, as a target of miR-23b. Expression of miR-23b decreased Sp1 mRNA levels via 3’UTR binding, as assessed in luciferase reporter assays. On the other hand, genetic and/or pharmacological inhibition of Sp1 led to miR-23b upregulation, thus highlighting the occurrence of a feedback loop between miR-23b and its target. Of note, miR-23b transfection significantly reduced Sp1-driven NF-kB activity in MM and WM cells. Finally, c-Myc, an important oncogenic transcription factor known to stimulate MM cell proliferation, has been shown to transcriptionally repress miR-23b. Moreover, treatment with the demethylating agent 5-aza-deoxycitidine significantly increase the expression of miR-23b in MM1S and KMS-11 cells suggesting that promoter methylation may be an additional mechanism of miR-23b suppression in myeloma. Thus MYC-dependent miR-23b repression in myeloma cells may allow activation of oncogenic transcription factors Sp1 and NF-κB, representing the first feed forward loop with critical growth and survival role in myeloma. Taken together, these data support a model in which the humoral environment reduces miR-23b expression in tumor cells, suggesting a tumor suppressor role in MM and WM and highlighting the potential of a miR-23b-based replacement therapy to treat these hematologic malignancies. Disclosures: Anderson: gilead: Consultancy; onyx: Consultancy; celgene: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership.

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