The LIM-only protein FHL2 reduces vascular lesion formation involving inhibition of proliferation and migration of smooth muscle cells

The LIM-only protein FHL2, also known as DRAL or SLIM3, has a function in fine-tuning multiple physiological processes. FHL2 is expressed in the vessel wall in smooth muscle cells (SMCs) and endothelial cells and conflicting data have been reported on the regulatory function of FHL2 in SMC phenotype transition. At present the function of FHL2 in SMCs in vascular injury is unknown. Therefore, we studied the role of FHL2 in SMC-rich lesion formation. In response to carotid artery ligation FHL2-deficient (FHL2-KO) mice showed accelerated lesion formation with enhanced Ki67 expression compared with wild-type (WT)-mice. Consistent with these findings, cultured SMCs from FHL2-KO mice showed increased proliferation through enhanced phosphorylation of extracellular-regulated kinase-1/2 (ERK1/2) and induction of CyclinD1 expression. Overexpression of FHL2 in SMCs inhibited CyclinD1 expression and CyclinD1-knockdown blocked the enhanced proliferation of FHL2-KO SMCs. We also observed increased CyclinD1 promoter activity in FHL2-KO SMCs, which was reduced upon ERK1/2 inhibition. Furthermore, FHL2-KO SMCs showed enhanced migration compared with WT SMCs. In conclusion, FHL2 deficiency in mice results in exacerbated SMC-rich lesion formation involving increased proliferation and migration of SMCs via enhanced activation of the ERK1/2-CyclinD1 signaling pathwa

Inhibition of GTPase Rac1 in endothelium by 6-mercaptopurine results in immunosuppression in nonimmune cells: new target for an old drug

Azathioprine and its metabolite 6-mercaptopurine (6-MP) are well established immunosuppressive drugs. Common understanding of their immunosuppressive properties is largely limited to immune cells. However, in this study, the mechanism underlying the protective role of 6-MP in endothelial cell activation is investigated. Because 6-MP and its derivative 6-thioguanosine-5'-triphosphate (6-T-GTP) were shown to block activation of GTPase Rac1 in T lymphocytes, we focused on Rac1-mediated processes in endothelial cells. Indeed, 6-MP and 6-T-GTP decreased Rac1 activation in endothelial cells. As a result, the compounds inhibited TNF-α-induced downstream signaling via JNK and reduced activation of transcription factors c-Jun, activating transcription factor-2 and, in addition, NF κ-light-chain-enhancer of activated B cells (NF-κB), which led to decreased transcription of proinflammatory cytokines. Moreover, 6-MP and 6-T-GTP selectively decreased TNF-α-induced VCAM-1 but not ICAM-1 protein levels. Rac1-mediated generation of cell membrane protrusions, which form docking structures to capture leukocytes, also was reduced by 6-MP/6-T-GTP. Consequently, leukocyte transmigration was inhibited after 6-MP/6-T-GTP treatment. These data underscore the anti-inflammatory effect of 6-MP and 6-T-GTP on endothelial cells by blocking Rac1 activation. Our data provide mechanistic insight that supports development of novel Rac1-specific therapeutic approaches against chronic inflammatory disease

Nur77 variants solely comprising the amino-terminal domain activate hypoxia-inducible factor-1 α and affect bone marrow homeostasis in mice and humans

Gene targeting via homologous recombination can occasionally result in incomplete disruption of the targeted gene. Here, we show that a widely used Nur77-deficient transgenic mouse model expresses a truncated protein encoding for part of the N-terminal domain of nuclear receptor Nur77. This truncated Nur77 protein is absent in a newly developed Nur77-deficient mouse strain generated using Cre-Lox recombination. Comparison of these two mouse strains using immunohistochemistry, flow cytometry, and colony-forming assays shows that homologous recombination-derived Nur77-deficient mice, but not WTor Cre-Lox-derived Nur77-deficient mice, suffer from liver immune cell infiltrates, loss of splenic architecture, and increased numbers of bone marrow hematopoietic stem cells and splenic colony-forming cells with age. Mechanistically, we demonstrate that the truncated Nur77 N-terminal domain protein maintains the stability and activity of hypoxia-inducible factor (HIF)-1, a transcription factor known to regulate bone marrow homeostasis. Additionally, a previously discovered, but uncharacterized, human Nur77 transcript variant that encodes solely for its N-terminal domain, designated TR3β, can also stabilize and activate HIF-1α. Meta-analysis of publicly available microarray data sets shows that TR3β is highly expressed in human bone marrow cells and acute myeloid leukemia samples. In conclusion, our study provides evidence that a transgenic mouse model commonly used to study the biological function of Nur77 has several major drawbacks, while simultaneously identifying the importance of nongenomic Nur77 activity in the regulation of bone marrow homeostasis

FHL2 deficiency enhances SMC proliferation via activation of ERK1/2.

<p><b>A</b>, Serum-starved SMCs were stimulated with or without FCS and treated with or without PD98059 (ERK1/2 inhibitor, 25µM). Cells were pulse-labeled with BrdU to measure DNA synthesis. <b>B</b>, SMCs from WT and FHL2-KO were seeded at equal density. 1 day after seeding, cells were treated with or without PD98059 and cells were counted manually. <b>C–D</b>, Western blot analysis (C) and quantification (D) for pERK1/2 in serum-starved SMCs after overexpression with or with out FHL2 followed by FCS stimulation for the indicated time periods, showing enhanced and prolonged activation of ERK1/2 in FHL2-defeicient SMCs and reduced activation of ERK1/2 in FHL2-KO SMCs after overexpression of FHL2. Data represent means±SD. *<i>P</i><0.05 for FHL2-KO versus WT.</p

Deficiency of FHL2 accelerates neointima formation after carotid artery ligation.

<p><b>A, C</b> and <b>E</b>; Representative cross sections of hematoxylin/eosin-stained carotid arteries from WT and FHL2-KO mice ligated for 1 (A), 2 (C) and 4 weeks (E). <b>B, D</b> and <b>F</b>; Quantitative analysis of neointima/media ratio and neointimal area in histological sections from WT and FHL2-KO mice ligated for 1 (B), 2 (D) and 4 weeks (F), revealed increased lesion formation in FHL2-KO mice. n = 7 for 1 and 2 weeks and n = 14 for 4 weeks. Three consecutive sections per mouse at each location were employed in the analysis. Lesions were characterized at 1.7, 2.0 and 2.3 mm from the reference point at 1, 2 and 4 weeks, respectively. Values are mean±SEM. *<i>P</i><0.05 for FHL2-KO versus WT mice.</p

FHL2 regulates cell proliferation via modulation of CycinD1 expression.

<p><b>A</b>, SMCs were transduced with lentiviral particles encoding FHL2 and assayed for CyclinD1 mRNA expression, showing that FHL2 inhibits its expression. <b>B</b>, Serum-starved WT SMCs were transduced with lentiviral particles encoding shCtrl, shCyclinD1 #1 and shCyclinD1 #2 and were pulse-labeled with BrdU to measure DNA synthesis. <b>C</b>, The CyclinD1 promoter-reporter plasmid showed higher induction in FHL2-KO SMCs stimulated with FCS than in WT SMCs. The ERK1/2 inhibitor PD98059 partly reduces this induction. Data represent means±SD. *<i>P</i><0.05 for FHL2-KO versus WT.</p

FHL2-KO SMCs migrate faster.

<p><b>A</b>, A scratch was made in a confluent layer of serum-starved SMCs that were stimulated with PDGF (20 ng/ml). Images were captured every 10 min using a live cell microscope and representative images at 0, 16 and 32 h are shown. Movies of the movement are in the online supplement. <b>B</b>, Quantitative analysis of SMC migration in the scratch wound assay showing that FHL2-KO SMCs migrated 1.8 fold faster than WT SMCs. <b>C</b>, SMCs were treated with or without PD98059 and cell migration was evaluated using a trans-well assay. Cells were labeled with a fluorescent dye and seeded in the upper chamber. Cell migration was measured as fluorescence after 3 h. <b>D</b>, SMC migration was evaluated using a trans-well assay after knock-down of FHL2 using lentiviral particles encoding shCtrl, shFHL2#1 and shFHL2#2 in WT SMCs. Cell migration was measured as fluorescence after 3 h. Data represent means±SD. *<i>P</i><0.05 for shCtrl versus shFHL2. <b>E</b>, Schematic representation of FHL2 function in the modulation of SMC-rich lesion formation. FHL2 modulates SMC-rich lesion formation by inhibiting proliferation and migration of SMCs via the ERK1/2-CyclinD1signaling pathway.</p

FHL2 deficiency enhances cell proliferation <i>in vivo</i>.

<p><b>A</b>, To assess the extent of proliferation in the vascular lesions, representative sections of injured carotid arteries from WT and FHL2-KO mice ligated for 1, 2 and 4 weeks were immunostained for Ki67. n = 7 for 1 and 2 weeks and n = 14 for 4 weeks. <b>B–C</b>, qRT-PCR was performed to assess mRNA expression of Ki67 (B) and PCNA (C) in the ligated vessels from WT and FHL2-KO mice for the indicated time periods. Data are means±SD. *<i>P</i><0.05 for FHL2-KO versus WT mice.</p