Thymosin β4 induces the synthesis of plasminogen activator inhibitor 1 in cultured endothelial cells and increases its extracellular expression

Al‐Nedawi, Khalid; Czyż, Małgorzata; Bednarek, Radosław; Szemraj, Janusz; Świątkowska, Maria; Cierniewska-Cieslak, Aleksandra; Wyczółkowska, J; Cierniewski, Czesław S.

doi:10.1182/blood-2003-04-1015

Cited by 39 publications

(37 citation statements)

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“…In our studies of the biological activity of T␤4 we found that this 43-amino acid peptide strongly activates endothelial cells to express and release plasminogen activator inhibitor type 1 (PAI-1). It occurs via the mechanism involving the signaling pathway leading to activation of the MAPK cascade and enhanced c-Fos/c-Jun DNA binding activity (4). Such a role of AP-1 in activation of transcriptional events by T␤4 was confirmed by independent studies (5).…”

supporting

confidence: 56%

Binding of PAI-1 to Endothelial Cells Stimulated by Thymosin β4 and Modulation of Their Fibrinolytic Potential

Boncela

Smolarczyk

Wyroba

et al. 2006

Journal of Biological Chemistry

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Our previous studies showed that thymosin ␤4 (T␤4) induced the synthesis of plasminogen activator inhibitor-1 (PAI-1) in cultured human umbilical vein endothelial cells (HUVECs) via the AP-1 dependent mechanism and its enhanced secretion. In this work we provide evidence that the released PAI-1 is accumulated on the surface of HUVECs, exclusively in its active form, in a complex with ␣1-acid glycoprotein (AGP) that is also up-regulated and released from the cells. This mechanism is supported by several lines of experiments, in which expression of both proteins was analyzed by flow cytometry and their colocalization supported by confocal microscopy. PAI-1 did not bind to quiescent cells but only to the T␤4-activated endothelial cells. In contrast, significant amounts of AGP were found to be associated with the cells overexpressing enhanced green fluorescent protein (EGFP)-␣1-acid glycoprotein (AGP) without T␤4 treatment. The AGP⅐PAI-1 complex was accumulated essentially at the basal surface of endothelial cells, and such cells showed (a) morphology characteristic for strongly adhered and spread cells and (b) significantly reduced plasmin formation. Taken together, these results provide the evidence supporting a novel mechanism by which active PAI-1 can be bound to the T␤4-activated endothelial cells, thus influencing their adhesive properties as well as their ability to generate plasmin.Thymosin ␤4 (T␤4) 2 promotes skin and corneal wound healing through its effects on cell migration, angiogenesis and possibly cell survival (1-3). However, the precise molecular mechanism through which it functions and its potential role in solid organ wound healing remains unknown. In our studies of the biological activity of T␤4 we found that this 43-amino acid peptide strongly activates endothelial cells to express and release plasminogen activator inhibitor type 1 (PAI-1). It occurs via the mechanism involving the signaling pathway leading to activation of the MAPK cascade and enhanced c-Fos/c-Jun DNA binding activity (4). Such a role of AP-1 in activation of transcriptional events by T␤4 was confirmed by independent studies (5). Recently, T␤4 was reported to stimulate migration of cardiomyocytes and endothelial cells and promote survival of cardiomyocytes by formation of a functional complex with PINCH and integrin-linked kinase, resulting in activation of the survival kinase Akt (6). These observations revealed that T␤4 affects cellular functions by activation of several signaling pathways and induces changes in the cell properties necessary for both migration and survival. Here, we show that T␤4 up-regulates expression and induces the surface accumulation of ␣1-acid glycoprotein (AGP) known to bind PAI-1 and prolong its inhibitory activity (7). AGP is expressed by activated endothelial cells and exerts its effect by interacting with components of the endothelial glycocalyx (8). In this study, we identify a previously unrecognized function of AGP, a direct and primary role in plasminogen activation. We provide evidence that t...

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supporting

confidence: 56%

Binding of PAI-1 to Endothelial Cells Stimulated by Thymosin β4 and Modulation of Their Fibrinolytic Potential

Boncela

Smolarczyk

Wyroba

et al. 2006

Journal of Biological Chemistry

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“…Thymosin b4 activates various signaling pathways including integrin-linked kinase (ILK), Jnk-1, extracellular signal-regulated kinase (Erk), and c-Jun. 14,16) In our study, pseudopodia formation was significantly inhibited by a PI3K inhibitor in thymosin b4 overexpressing LNCaP cells. ILK activity is positively regulated by PI3K, which leads to Cdc42 and Rac activation, then pseudopodia formation.…”

Section: Discussionmentioning

confidence: 89%

Overexpression of Thymosin .BETA.4 Increases Pseudopodia Formation in LNCaP Prostate Cancer Cells

Ito

Iguchi

Usui

et al. 2009

Biological & Pharmaceutical Bulletin

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Most patients with advanced prostate cancer are candidates for androgen ablation therapy because growth and progression of prostate cancer cells are initially androgen dependent.1,2) Although androgen ablation therapy initially causes tumor regression in Ͼ80% of cases, prostate cancer eventually progresses from an androgen-dependent to an aggressive androgen-independent state after the therapy, and the prostate cancer in this state is often difficult to cure. 2,3) One of the reasons why prostate cancer cells after androgen ablation therapy acquire aggressive phenotype is changes occur in the expression of androgen-regulated genes after the therapy. 4,5) In our recent study, we found that thymosin b4 expression in androgen-sensitive prostate cancer LNCaP cells is elevated in androgen-deprived culture condition. 6)Thymosin b4 is a small acidic 4.9-kDa polypeptide widely distributed in human tissues. Thymosin b4 functions as a major G-actin sequestering factor that modulates dynamic changes of actin cytoskeleton. 7) In addition, thymosin b4 is involved in a variety of physiologic and pathologic processes. For example, thymosin b4 is known to promote wound healing, tumor metastasis, and angiogenesis. [8][9][10] Overexpression of thymosin b4 is detected highly in metastatic cancer cells in human and animal models. [11][12][13] In addition, thymosin b4 stimulates cardiac cell migration and survival related to cardiac repair.14) In the present study, we found that thymosin b4 expression increases pseudopodia formation related to cell migration and invasion in human prostate LNCaP cells. MATERIALS AND METHODS MaterialsWortmannin was purchased from SigmaAldrich Japan K.K. (Tokyo, Japan). Toxin B from Clostridium difficile was from List Biological Laboratories (Campbell, CA, U.S.A.). U0126 was purchased from Wako Pure Chemical (Osaka, Japan). Rapamycin was from Merck Ltd. (Tokyo, Japan).Cell Culture Human prostate cancer LNCaP cells were from American Type Culture Collection (Rockville, MD, U.S.A.). LNCaP cells were cultured in RPMI-1640 medium containing 10% fetal calf serum (FCS) under a humidified atmosphere with 5% CO 2 at 37°C. Plasmid ConstructionsTotal RNA from LNCaP cells was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, U.S.A.) then first-strand complementary DNA was synthesized from the total RNA using SuperScript III (Invitrogen) according to the manufacturer's instructions. The cDNA coding region of human thymosin b4 was amplified by polymerase chain reaction (PCR) from the cDNA using primers containing restriction sites for XbaI and HindIII. The sequences of the primers were, sense: 5Ј-GCTCTAGAAT-GTCTGACAAACCCGATATG-3Ј; antisense: 5Ј-CCAAGC-TTTTACGATTCGCCTGCTTGCTT-3Ј. PCR was performed using PrimeSTAR HS DNA Polymerase (Takara Bio Inc., Otsu, Japan) under the following conditions: 32 cycles of 30 s at 98°C, 30 s at 58°C, and 30 s at 72°C. The resulting PCR product was digested with XbaI and HindIII and inserted into the XbaI and HindIII sites of a pRK5 mammalian expression vector. The plasmid vect...

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“…Of relevance to sclerosis, exogenous thymosin ␤4 increases PAI-1 expression at both the mRNA and protein levels in endothelial cells (26). PAI-1 inhibits tissue-type plasminogen activator and urokinase-type plasminogen activator, preventing the activation of plasminogen to plasmin.…”

Section: Discussionmentioning

confidence: 99%

“…The last results in part is from Ang II induction of PAI-1 expression in vivo and in vitro (14,25). Furthermore, application of synthetic thymosin ␤4 increases PAI-1 expression in endothelial cells (26). We therefore assessed whether endogenous thymosin ␤4 affected PAI-1 expression in response to Ang II.…”

Section: Thymosin ␤4 and Sclerosis Mechanismsmentioning

confidence: 99%

Proteomic Patterns and Prediction of Glomerulosclerosis and Its Mechanisms

Shyr

Liang³

et al. 2005

Journal of the American Society of Nephrology

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Protein expression profiles linked to sclerosis in the 5/6 nephrectomy (Nx) rat model of focal segmental glomerulosclerosis were investigated. Sections of control glomeruli from normal baseline Nx tissue and nonsclerotic and sclerotic glomeruli from 12 wk after 5/6 Nx were isolated by laser capture microdissection. Protein profiles were acquired directly by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Classification accuracy was 99.2% for distinguishing normal versus sclerotic glomeruli and 96.7 and 97.8% for nonsclerotic versus normal and sclerotic glomeruli, respectively. The proteomic pattern of the nonsclerotic glomeruli was more similar to sclerotic than normal glomeruli (P < 0.0001). Thymosin ␤4, a protein with relevant interactions with plasminogen activator inhibitor-1, angiogenesis, and wound healing, was identified as a key differentially expressed protein. P rogression of focal segmental glomerulosclerosis (FSGS) from early injury to overt sclerosis involves injury to all glomerular cell types (1,2), through multiple complex mechanisms. With the advancement of proteomic techniques (3,4), simultaneous examination of hundreds of proteins related to kidney disease holds promise in unraveling novel underlying mechanisms of progression and thus identifying possible targets for intervention in FSGS.To obtain protein expression profiles from a localized area, laser capture microdissection (LCM) has been combined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) for tissue protein profiling (5,6). LCM is an important tool in biologic research, enabling the isolation of specific cell populations from a heterogeneous tissue section (7). The technique of direct protein profiling from the laser capture microdissected sample using MALDI MS is fast, sensitive, and accurate. This technique has been applied in several studies, including those of human breast carcinoma (5), mouse epididymis (8), and human lung carcinoma (9). This technique enables the determination of protein expression profiles from even minute tissue structures within limited samples.The focal segmental nature of sclerosis in FSGS raises the question of whether at a given time point the remaining nonsclerotic glomeruli are already programmed to sclerotic pathways or, alternatively, whether these remaining nonsclerotic glomeruli have less prosclerotic activation and thus may be more susceptible to therapy. We therefore sought to examine proteomic profiles from the nonsclerotic glomeruli in FSGS and compare profiles from them with normal glomeruli and glomeruli with established sclerosis. We aimed first to establish the sensitivity of LCM and MALDI MS techniques to differentiate sclerosis from normal and, second, to investigate whether nonsclerotic glomeruli have a prosclerotic phenotype at the protein level. We also sought to identify key differentially expressed protein markers to advance our understanding of mechanisms and possible intervention in progressive renal disease. Materials and ...

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Thymosin β4 induces the synthesis of plasminogen activator inhibitor 1 in cultured endothelial cells and increases its extracellular expression

Cited by 39 publications

References 28 publications

Binding of PAI-1 to Endothelial Cells Stimulated by Thymosin β4 and Modulation of Their Fibrinolytic Potential

Binding of PAI-1 to Endothelial Cells Stimulated by Thymosin β4 and Modulation of Their Fibrinolytic Potential

Overexpression of Thymosin .BETA.4 Increases Pseudopodia Formation in LNCaP Prostate Cancer Cells

Proteomic Patterns and Prediction of Glomerulosclerosis and Its Mechanisms

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