Synovial fluid of patients with rheumatoid arthritis induces ��-smooth muscle actin in human adipose tissue-derived mesenchymal stem cells through a TGF-��1-dependent mechanism

Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disorder that causes the immune system to attack the joints. Transforming growth factor-β1 (TGF-β1) is a secreted protein that promotes differentiation of synovial fibroblasts to α-smooth muscle actin (α-SMA)-positive myofibroblasts to repair the damaged joints. Synovial fluid from patients with RA (RA-SF) induced expression of α-SMA in human adipose tissue-derived mesenchymal stem cells (hASCs). RA-SF-induced α-SMA expression was abrogated by immunodepletion of TGF-β1 from RA-SF with anti-TGF-β1 antibody. Furthermore, pretreatment of hASCs with the TGF-β type I receptor inhibitor SB431542 or lentiviral small hairpin RNA-mediated silencing of TGF-β type I receptor expression in hASCs blocked RA-SF-induced α-SMA expression. Small interfering RNA-mediated silencing of Smad2 or adenoviral overexpression of Smad7 (an inhibitory Smad isoform) completely inhibited RA-SF-stimulated α-SMA expression. These results suggest that TGF-β1 plays a pivotal role in RA-SF-induced differentiation of hASCs to α-SMA-positive cells.     

Downstream components of RhoA required for signal pathway of superoxide formation during phagocytosis of serum opsonized zymosans in macrophages

Rac1 and Rac2 are essential for the control of oxidative burst catalyzed by NADPH oxidase. It was also documented that Rho is associated with the superoxide burst reaction during phagocytosis of serum- (SOZ) and IgG-opsonized zymosan particles (IOZ). In this study, we attempted to reveal the signal pathway components in the superoxide formation regulated by Rho GTPase. Tat-C3 blocked superoxide production, suggesting that RhoA is essentially involved in superoxide formation during phagocytosis of SOZ. Conversely SOZ activated both RhoA and Rac1/2. Inhibition of RhoA-activated kinase (ROCK), an important downstream effector of RhoA, by Y27632 and myosin light chain kinase (MLCK) by ML-7 abrogated superoxide production by SOZ. Extracellular signaling-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) were activated during phagocytosis of SOZ, and Tat-C3 and SB203580 reduced ERK1/2 and p38 MAPK activation, suggesting that RhoA and p38 MAPK may be upstream regulators of ERK1/2. Inhibition of ERK1/2, p38 MAPK, phosphatidyl inositol 3-kinase did not block translocation of RhoA to membranes, suggesting that RhoA is upstream to these kinases. Inhibition of RhoA by Tat-C3 blocked phosphorylation of p47(PHOX). Taken together, RhoA, ROCK, p38MAPK, ERK1/2, and p47(PHOX) may be subsequently activated, leading to activation of NADPH oxidase to produce superoxide.     

CXCR4 inhibition suppresses Cd-induced renal oxidative stress,apoptosis, and fibrosis by inhibiting the TGF-β1/Smad pathway

This study aimed to investigate the role and mechanism of CXC chemokine receptor 4 (CXCR4) in cadmium (Cd)-induced renal injury. CXCR4 and TGF-β1/Smad pathway protein levels were detected by western blotting. Indicators related to renal function and oxidative stress factors were assessed and reactive oxygen species (ROS) level was evaluated by staining. TUNEL was used to measure apoptosis rate. PAS and Masson's trichrome staining were used to detect the level of renal fibrosis. The expression of Bcl-2, Bax, Cleaved-caspase 3, fibronectin, and collagen I proteins were detected by western blotting, immunohistochemistry, or immunofluorescence. The expression of CXCR4 was increased in a Cd-induced chronic renal injury model in rats. Si-CXCR4 decreased levels of TGF-β1, TGF-βR1, p-Smad2/Smad2, p-Smad3/Smad3, the renal weight index, urine protein, blood urea nitrogen, blood creatinine, and levels of MDA but raised the levels of SOD and GSH-Px. In addition, si-CXCR4 inhibited apoptosis in Cd-treated rats. CXCR4 inhibition alleviated fibrosis levels in Cd-treated rats. In Cd-treated cells, TGF-β attenuated the suppressive effect of CXCR4 inhibition on the TGF-β1/Smad pathway. TGF-β intervention increased MDA and ROS, and downregulated SOD and GSH-Px. TGF-β attenuated the inhibitory effect of CXCR4 on apoptosis and fibrosis. CXCR4 inhibition decreased levels of Cd-induced renal oxidative stress, apoptosis, and fibrosis by inhibiting the TGF-β1/Smad pathway.     

AMPA, not NMDA, activates RhoA GTPases and subsequently phosphorylates moesin

Glutamate induced rapid phosphorylation of moesin, one of ERM family proteins involved in the ligation of membrane to actin cytoskeleton, in rat hippocampal cells (JBC, 277:16576-16584, 2002). However, the identity of glutamate receptor has not been explored. Here we show that a-amino- 3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor is responsible for glutamate-induced RhoA activation and phosphorylation of moesin. Glutamate induced phosphorylation at Thr-558 of moesin was still detectible upon chelation of Ca(2+), suggesting involvement of AMPA receptor instead of N-methyl D-Aspartate (NMDA) receptor in this phosphorylation of moesin. AMPA but not NMDA- induced moesin phosphorylation was independent of Ca(2+). Both AMPA and NMDA but not Kainate induced moesin phosphorylation at similar levels. However, the kinetics of phosphorylation varied greatly between AMPA and NMDA where AMPA treatment rapidly increased phosphomoesin, which reached a maximum at 10 min after treatment and returned to a basal level at 30 min. In contrast, NMDA-induced phosphorylation of moesin reached a maximum at 30 min after treatment and was remained at higher levels at 60 min. A possible involvement of RhoA and its downstream effector, Rho kinase in the AMPA receptor-triggered phosphorylation of moesin was also explored. The kinetics for the glutamate- induced membrane translocation of RhoA was similar to that of moesin phosphorylation induced by AMPA. Moreover, Y-27632, a specific Rho kinase inhibitor, completely blocked AMPA-induced moesin phosphorylation but had no effect on NMDA-induced moesin phosphorylation. These results suggest that glutamate-induced phosphorylation of moesin may be mediated through the AMPA receptor/RhoA/Rho kinase pathway.     

Thromboxane A2 modulates migration, proliferation, and differentiation of adipose tissue-derived mesenchymal stem cells

Prostanoid metabolites are key mediators in inflammatory responses, and accumulating evidence suggests that mesenchymal stem cells (MSCs) can be recruited to injured or inflamed tissues. In the present study, we investigated whether prostanoid metabolites can regulate migration, proliferation, and differentiation potentials of MSCs. We demonstrated herein that the stable thromboxane A₂ (TxA₂) mimetic U46619 strongly stimulated migration and proliferation of human adipose tissue-derived MSCs (hADSCs). Furthermore, U46619 treatment increased expression of α-smooth muscle actin (α-SMA), a smooth muscle marker, in hADSCs, suggesting differentiation of hADSCs into smooth muscle-like cells. U46619 activated ERK and p38 MAPK, and pretreatment of the cells with the MEK inhibitor U0126 or the p38 MAPK inhibitor SB202190 abrogated the U46619-induced migration, proliferation, and α-SMA expression. These results suggest that TxA₂ plays a key role in the migration, proliferation, and differentiation of hADSCs into smooth muscle-like cells through signaling mechanisms involving ERK and p38 MAPK.     

TGF-β-signaling with small molecule FKBP12 antagonists that bind myristoylated FKBP12-TGF-β type I receptor fusion proteins

Background: Growth arrest in many cell types is triggered by transforming growth factor beta (TGF-β), which signals through two TGF-β receptors (type I, TGF-βRI, and type II, TGF-βRII). In the signaling pathway, TGF-β binds to the extracellular domain of TGF-βRII, which can then transphosphorylate TGF-βRI in its glycine/serine (GS)-rich box. Activated TGF-βRI phosphorylates two downstream effectors, Smad2 and Smad3, leading to their translocation into the nucleus. Cell growth is arrested and plasminogen activator inhibitor 1 (PAI-1) is upregulated. We investigated the role of the immunophilin FKBP12, which can bind to the GS box of TGF-βRI, in TGF-β signaling.Results: Overexpression of myristoylated TGF-βRI and TGF-βRII cytoplasmic tails caused constitutive nuclear translocation of a green-fluorescent-protein-Smad2 construct in COS-1 cells, and constitutive activation of a PAI-1 reporter plasmid in mink lung cells. Fusing FKBP12 to TGF-βRI resulted in repression of autosignaling that could be alleviated by FK506M or rapamycin (two small molecules that can bind to FKBP12). Mutation of the FKBP12-binding site in the FKBP12-TGF-βRI fusion protein restored constitutive signaling. An acidic mutation in the FKBP12-TGFβRI protein allowed FKBP12 antagonists to activate signaling in the absence of TGF-βRII. Further mutations in the TGF-βRI FKBP12-binding site resulted in TGF-β signaling that was independent of both TGF-βRII and FKBP12 antagonists.Conclusions: Fusing FKBP12 to TGF-βRI results in a novel receptor that is activated by small molecule FKBP12 antagonists. These results suggest that FKBP12 binding to TGF-βRI is inhibitory and that FKBP12 plays a role in inhibiting TGF-β superfamily signals.     

Orphan nuclear receptor small heterodimer partner inhibits angiotensin II-stimulated PAI-1 expression in vascular smooth muscle cells

Angiotensin II is a major effector molecule in the development of cardiovascular disease. In vascular smooth muscle cells (VSMCs), angiotensin II promotes cellular proliferation and extracellular matrix accumulation through the upregulation of plasminogen activator inhibitor-1 (PAI-1) expression. Previously, we demonstrated that small heterodimer partner (SHP) represses PAI-1 expression in the liver through the inhibition of TGF-β signaling pathways. Here, we investigated whether SHP inhibited angiotensin II-stimulated PAI-1 expression in VSMCs. Adenovirus-mediated overexpression of SHP (Ad-SHP) in VSMCs inhibited angiotensin II- and TGF-β-stimulated PAI-1 expression. Ad-SHP also inhibited angiotensin II-, TGF-β- and Smad3-stimulated PAI-1 promoter activity, and angiotensin II-stimulated AP-1 activity. The level of PAI-1 expression was significantly higher in VSMCs of SHP^-/- mice than wild type mice. Moreover, loss of SHP increased PAI-1 mRNA expression after angiotensin II treatment. These results suggest that SHP inhibits PAI-1 expression in VSMCs through the suppression of TGF-β/Smad3 and AP-1 activity. Thus, agents that target the induction of SHP expression in VSMCs might help prevent the development and progression of atherosclerosis.     

Statin inhibits interferon-gamma-induced expression of intercellular adhesion molecule-1 (ICAM-1) in vascular endothelial and smooth muscle cells

Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, known as statins, are widely used for primary and secondary prevention of coronary artery atherosclerosis. Pathogenesis of atherosclerosis is multistep processes where transendothelial migration of various leukocytes including monocytes is a crucial step. Interferon-gamma (IFN-gamma) contributes in this process by activating macrophages and T-lymphocytes, and by inducing adhesion molecules in vascular endothelial and smooth muscle cells. In this study we investigated the expression of intercellular cell adhesion molecule-1 (ICAM-1) in transformed endothelial cell line ECV304 cells as influenced by lovastatin, tumor necrosis factor-alpha (TNF-alpha) and IFN-gamma. Results show that lovastatin suppresses expression of ICAM-1 by inhibiting the IFN-gamma-induced extracellular signal-regulated kinase (ERK) p44/p42-STAT1 signaling pathway. In cells treated with lovastatin and IFN-gamma, ICAM-1 was expressed at a lower level than in cells treated with IFN-gamma alone. However, lovastatin does not reduce TNF-alpha induced expression of ICAM-1. A similar result was observed in cells treated with the MEKK inhibitor PD98059 and IFN-gamma. Cis-acting DNA sequence elements were identified in the 5'-flanking region of the ICAM-1 promoter that mediate inhibition by lovastatin; these sequences map to the IFN-gamma activated site which also binds the STAT1 homodimer. However, lovastatin did not inhibit IFN-gamma-mediated induction of the Y701 phosphorylated form of STAT1. But lovastatin does inhibit the IFN-gamma-mediated phosphorylation of ERK1/ERK2 (T202/Y204) and S727 phosphorylation of STAT1. TNF-alpha does not induce phosphorylation of ERK1/ERK2 and S727 in ECV304 and smooth muscle cells. The results provide the evidences that statins may have beneficial effects by inhibiting IFN-gamma action in atherosclerotic process     

Inhibitory effect of receptor for advanced glycation end products (RAGE) on the TGF-β-induced alveolar epithelial to mesenchymal transition

Idiopathic pulmonary fibrosis (IPF) is a lethal parenchymal lung disease characterized by myofibroblast proliferation. Alveolar epithelial cells (AECs) are thought to produce myofibroblasts through the epithelial to mesenchymal transition (EMT). Receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface receptors whose activation is associated with renal fibrosis during diabetes and liver fibrosis. RAGE is expressed at low basal levels in most adult tissues except the lung. In this study, we evaluated the interaction of ligand advanced glycation end products (AGE) with RAGE during the epithelial to myofibroblast transition in rat AECs. Our results indicate that AGE inhibited the TGF-β-dependent alveolar EMT by increasing Smad7 expression, and that the effect was abolished by RAGE siRNA treatment. Thus, the induction of Smad7 by the AGE-RAGE interaction limits the development of pulmonary fibrosis by inhibiting TGF-β-dependent signaling in AECs.     

Enhanced integrin mediated signaling and cell cycle progression on fibronectin mimetic peptide amphiphile monolayers

In recent years, a variety of biomimetic constructs have emerged which mimic the bioactive sequences found in the natural extracellular matrix (ECM) proteins such as fibronectin (FN) that promote cell adhesion as well as proliferation on artificially functionalized interfaces. Much interest lies in investigating the ability of the ECM mimetic materials in regulating a number of vital cell functions including differentiation, gene expression, migration, and proliferation. A peptide amphiphile PR_b containing both the cell adhesive GRGDSP and synergistic PHSRN peptide sequences was developed in our group that was shown to support enhanced cell proliferation and ECM FN secretion as compared to GRGDSP and FN functionalized interfaces. In this study, we have investigated the binding affinity of the PR_b peptide ligand with the FN cell surface receptor, the α(5)β(1) integrin. We compared PR_b functionalized surfaces with FN and BSA coated surfaces and GRGDSP functionalized surfaces in terms of promoting intracellular signaling cascades that are essential for enhanced cellular activity. Specifically, we studied the phosphorylation of focal adhesion kinase (FAK) at tyrosine residues Y397 and Y576 and the formation of cyclin D1, both of which are intracellular markers of integrin mediated attachment of cells, signaling pathways, and progression of cell cycle. FAK and cyclin D1 encourage enhanced cell proliferation, differentiation, and gene expression. Our results show that the PR_b peptide ligand has a specific and strong binding affinity for the α(5)β(1) integrin with a dissociation constant of 76.3 ± 6.3 nM. The PR_b peptide ligands supported enhanced FAK phosphorylation activity and increased cyclin D1 formation as compared to the widely used GRGDSP ligand, the native protein FN (positive control), and BSA nonadhesive surfaces (negative control). These results encourage the use of the FN mimetic PR_b peptide in functionalizing biomaterials for potential tissue engineering and therapeutic applications.     

Simvastatin inhibits induction of matrix metalloproteinase-9 in rat alveolar macrophages exposed to cigarette smoke extract

Matrix metalloproteinase-9 (MMP-9) may play an important role in emphysematous change in chronic obstructive pulmonary disease (COPD), one of the leading causes of mortality and morbidity worldwide. We previously reported that simvastatin, an inhibitor of HMG-CoA reductase, attenuates emphysematous change and MMP-9 induction in the lungs of rats exposed to cigarette smoke. However, it remained uncertain how cigarette smoke induced MMP-9 and how simvastatin inhibited cigarette smoke-induced MMP-9 expression in alveolar macrophages (AMs), a major source of MMP-9 in the lungs of COPD patients. Presently, we examined the related signaling for MMP-9 induction and the inhibitory mechanism of simvastatin on MMP-9 induction in AMs exposed to cigarette smoke extract (CSE). In isolated rat AMs, CSE induced MMP-9 expression and phosphorylation of ERK and Akt. A chemical inhibitor of MEK1/2 or PI3K reduced phosphorylation of ERK or Akt, respectively, and also inhibited CSE-mediated MMP-9 induction. Simvastatin reduced CSE-mediated MMP-9 induction, and simvastatin-mediated inhibition was reversed by farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP). Similar to simvastatin, inhibition of FPP transferase or GGPP transferase suppressed CSE-mediated MMP-9 induction. Simvastatin attenuated CSE-mediated activation of RAS and phosphorylation of ERK, Akt, p65, IκB, and nuclear AP-1 or NF-κB activity. Taken together, these results suggest that simvastatin may inhibit CSE-mediated MMP-9 induction, primarily by blocking prenylation of RAS in the signaling pathways, in which Raf-MEK-ERK, PI3K/Akt, AP-1, and IκB-NF-κB are involved.     

A new synthetic chalcone derivative, 2-hydroxy-3',5,5'-trimethoxychalcone (DK-139), suppresses the Toll-like receptor 4-mediated inflammatory response through inhibition of the Akt/NF-κB pathway in BV2 microglial cells

Microglial cells are the resident innate immune cells that sense pathogens and tissue injury in the central nervous system (CNS). Microglial activation is critical for neuroinflammatory responses. The synthetic compound 2-hydroxy-3',5,5'-trimethoxychalcone (DK-139) is a novel chalcone-derived compound. In this study, we investigated the effects of DK-139 on Toll-like receptor 4 (TLR4)-mediated inflammatory responses in BV2 microglial cells. DK-139 inhibited lipopolysaccharide (LPS)-induced TLR4 activity, as determined using a cell-based assay. DK-139 blocked LPS-induced phosphorylation of IκB and p65/RelA NF-κB, resulting in inhibition of the nuclear translocation and trans-acting activity of NF-κB in BV2 microglial cells. We also found that DK-139 reduced the expression of NF-κB target genes, such as those for COX-2, iNOS, and IL-1β, in LPS-stimulated BV2 microglial cells. Interestingly, DK-139 blocked LPS-induced Akt phosphorylation. Inhibition of Akt abrogated LPS-induced phosphorylation of p65/RelA, while overexpression of dominant- active p110CAAX enhanced p65/RelA phosphorylation as well as iNOS and COX2 expression. These results suggest that DK-139 exerts an anti-inflammatory effect on microglial cells by inhibiting the Akt/IκB kinase (IKK)/NF-κB signaling pathway.     

CD98 activation increases surface expression and clusteringof beta1 integrins in MCF-7 cells through FAK/Src- and cytoskeleton-independent mechanisms

CD98, a disulfide-linked 125-kDa heterodimeric type II transmembrane glycoprotein, regulates beta1 integrin- mediated cell adhesion. However, the molecular mechanisms underlying CD98-mediated activation of beta1 integrin are presently unclear. In this study, the effects of CD98 signaling on the expression and clustering of beta1 integrin were investigated. Activation of CD98 augmented surface expression of beta1 integrin on MCF-7 cells. Cross-linking CD98 induced clustering of beta1 integrins. Inhibition of phosphorylation of focal adhesion kimase (FAK) by PP2, an inhibitor of Src family kinase, reduced cell-extracellular matrix adhesion, but not surface expression and clustering of beta1 integrin on MCF-7 cells. This result was confirmed by over-expression of dominant negative forms of FAK. In addition, phalloidin or cytochalasin D inhibited CD98-mediated induction of cell-ECM adhesion, but not surface expression and clustering of beta1 integrins. The inhibitory effects of PP2, cytochalasin D or phalloidin on CD98-stimulated cell adhesion were diminished by pretreatment of cells with Mn2+, which is shown to induce conformational change of integrins. These results provide the first evidence that CD98 activation increases not only beta1 integrin affinity but also its surface expression and clustering and the latter is independent of FAK/Src and cytoskeleton.     

980 nm Photobiomodulation Promotes Osteo/Odontogenic Differentiation of the Stem Cells from Human Exfoliated Deciduous Teeth Via the Cross talk Between BMP/Smad and Wnt/β-Catenin Signaling Pathways

Increasing evidence suggests stem cells from human exfoliated deciduous teeth (SHEDs) serve as desirable sources of dentin regeneration. Photobiomodulation (PBM) has shown great potential in enhancing the proliferation and osteogenesis of human bone marrow mesenchymal stem cells (hBMMSCs). However, the specific role of PBM in odontogenic differentiation of SHEDs is little know, and we further investigated potential mechanism of PBM osteo/odontogenisis. A 980 nm diode laser with different energy densities of (0.5, 5, 10 J cm⁻²) in a 100-mW continuous wave was used for irradiation every 24 h. Osteo/odontogenic differentiation of SHEDs was achieved by performing alkaline phosphatase (ALP) and alizarin red staining (ARS) and osteo/odontogenic markers were also evaluated by qRT-PCR and western blotting. Additionally, western blot and immunohistochemical staining were performed to evaluate the levels of BMP/Smad and Wnt/β-catenin signaling-related proteins. We found that PBM at 5 J cm⁻¹ increased mineral deposition and upregulated the expression of related osteo/odontogenic markers along with the elevated expression of β-catenin and phosphorylation level of Smad1/5/9. Furthermore, Wnt signaling inhibition using DKK1 and BMP signaling inhibition using noggin inhibited PBM-induced osteo/odontogenic marker expression when used individually or jointly. In conclusion, PBM induces the osteo/odontogenic differentiation of SHEDs through cross talk between BMP/Smad and Wnt/β-catenin signaling pathways.     

Isoliquiritigenin Enhances the Beige Adipocyte Potential of Adipose-Derived Stem Cells by JNK Inhibition

Human adipose-derived stem cells (hASCs) can be isolated from fat tissue and have attracted interest for their potential therapeutic applications in metabolic disease. hASCs can be induced to undergo adipogenic differentiation in vitro by exposure to chemical agents or inductive growth factors. We investigated the effects and mechanism of differentiating hASC-derived white adipocytes into functional beige and brown adipocytes with isoliquiritigenin (ILG) treatment. Here, we showed that hASC-derived white adipocytes could promote brown adipogenesis by expressing both uncoupling protein 1 (UCP1) and PR/SET Domain 16 (PRDM16) following low-dose ILG treatments. ILG treatment of white adipocytes enhanced the expression of brown fat-specific markers, while the expression levels of c-Jun N-terminal kinase (JNK) signaling pathway proteins were downregulated. Furthermore, we showed that the inhibition of JNK phosphorylation contributed to white adipocyte differentiation into beige adipocytes, which was validated by the use of SP600125. We identified distinct regulatory effects of ILG dose responses and suggested that low-dose ILG induced the beige adipocyte potential of hASCs via JNK inhibition.