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FJ EXPRESS SUMMARY ARTICLE
The Full-length version of this article is also available, published online June 3, 2003 as doi:10.1096/fj.02-0785fje.
Published as doi: 10.1096/fj.02-0785fje.
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(The FASEB Journal. 2003;17:1529-1531.)
© 2003 FASEB

Arg-Gly-Asp-Ser (RGDS) peptide stimulates transforming growth factor ß1 transcription and secretion through integrin activation 1

R. ORTEGA-VELÁZQUEZ, M. L. DÍEZ-MARQUÉS, M. P. RUIZ-TORRES, M. GONZÁLEZ-RUBIO, M. RODRÍGUEZ-PUYOL and D. RODRÍGUEZ PUYOL2

Departments of Physiology and Medicine, Alcalá University, Nephrology Section, Hospital Príncipe de Asturias, and IRSIN, Madrid, Spain

2Correspondence: Departamento de Fisiología, Facultad de Medicina, Universidad de Alcalá, Carretera de Barcelona, Km 33,600, Alcalá de Henares, 28880 Madrid, Spain. E-mail: diego.rodriguez{at}uah.es

SPECIFIC AIMS

Through specific peptide motifs such as RGD (ARG-GLY-ASP), extracellular matrix (ECM) components interact with integrin and can modify the behavior of cells. We analyzed the effect of an RGD-containing peptide, RGDS, on the regulation of TGF-ß1 secretion by cultured human mesangial cells (HMC) and the intracellular mechanisms implied in signal transduction.

PRINCIPAL FINDINGS

1. RGDS stimulates TGF-ß1 secretion by cultured HMC
HMC were serum starved for 48 h and RGDS was added at different times (0–24 h) and concentrations (0–100 µM). The incubation medium was collected and the TGF-ß1 concentration was assessed by ELISA. HMC incubated with RGDS increased secretion of immunoreactive TGF-ß1 in a dose- and time-dependent manner. Maximal TGF-ß1 stimulation was twofold and occurred after 16 h of treatment with 100 µM RGDS. RGDS concentrations of >100 µM were also tested, but they induced significant cell detachment. RGES did not have any significant effect on TGF-ß1 secretion. The following experiments were performed with 50 µM RGDS for 16 h. Likewise, soluble fibronectin (2.5–10 µg/mL) produced an increase in TGF-ß1 synthesis in these cells.

2. RGDS stimulates the TGF-ß1 mRNA
RGDS (50 µM) induced a significant increase in the TGF-ß1 mRNA expression after 16 h that remained for 24 h as determined by Northern blot analysis. TGF-ß1 mRNA stability was determined with actinomycin D (1.6 mg/mL) treated HMC at 2, 4, and 6 h before RNA extraction. RGDS did not significantly modify the mRNA stability. On the other hand, TGF-ß1 promoter activity increased after 50 µM RGDS treatment, as measured by increased luciferase activity in HMC transfected with a plasmid containing the TGF-ß1 promoter linked to the luciferase reporter gene. This increased promoter activity was detected as early as 4 h after RGDS addition to cells, being maximal after 8 h. Thus, the increased steady-state levels of the TGF-ß1 mRNA were not the consequence of a higher stability of the transcript, but rather of an increase in promoter activity. No changes in promoter activity were detected with the same RGES concentration

3. RGDS effect is dependent on interactions with integrins
The role of the different integrins in the effects of RGDS was studied by using anti-integrin antibodies before RGDS incubation. HMC were serum starved for 48 h and pretreated with 30 µg/mL anti-integrin-{alpha}1, -{alpha}V, -ß1 antibody, and non-immune IgG for 4 h before the addition of 50 µM RGDS for 16 h. They were also treated for 16 h with 30 µ/mL of an anti-ß1 activating antibody. Anti-{alpha}V and anti-ß1, but not non-immune IgG or anti-{alpha}1, completely abolished the changes observed in TGF-ß1 synthesis after treatment of HMC with RGDS. On the other hand, the anti-ß1 activating antibody induced a significant increase of the immunoreactive TGF-ß1 production even in the absence of RGDS. Anti-{alpha}V and anti-ß1 antibodies also blocked the increased TGF-ß1 mRNA expression induced by RGDS (Fig. 1 ).



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  		Figure 1. RGDS activation of TGF-ß1 secretion and mRNA expression in human mesangial cells (HMC) is prevented by anti-integrin antibodies. A) Cells were incubated for 4 h with 30 µg/mL of non-immune IgG (Ig), anti-{alpha}1, anti-ß1, or anti-{alpha}v antibodies. 50 µM RGDS was added for 16 h and immunoreactive TGF-ß1 was measured in cell supernatants by ELISA. The same experiments were performed in the presence of 30 µg/mL of an anti-ß1 activator antibody (ß*) without RGDS. A RGE control is included. N = 7. B) Cells were incubated for 4 h with 30 µg/mL of anti-ß1 or anti-{alpha}v antibodies. Then 50 µM RGDS was added for 16 h and TGF-ß1 mRNA expression was measured in cells by Northern blot. Inset: A representative experiment. N = 6, *P < 0.05 vs. control (untreated cells).

4. RGDS increases the rate of tyrosine phosphorylation and integrin-linked kinase (ILK) activity
To test the intracellular mechanisms involved in the RGDS-dependent TGF-ß1 stimulation, the role of two major integrin-coupled pathways was evaluated. Tyrosine phosphorylation was evaluated by immunoblotting with an anti-phosphotyrosine antibody. Within 20 min, RGDS enhanced the tyrosine phosphorylation of different proteins. ILK activity was assayed in cell extracts by immunoprecipitation in vitro kinase assays using myelin basic protein as a substrate. ILK activity increased in cells treated with 50 µM RGDS, reaching a maximum after 30 min of incubation with RGDS.

5. Tyrosine kinase activity does not mediate the effect of RGDS on TGF-ß1 secretion
The role of tyrosine kinases in the RGDS responsiveness of HMC was determined in cells treated with 2 mM herbimycin A or 2 µg/mL genistein for 4 h. Tyrosine kinase inhibitors did not prevent the RGDS-dependent increase of TGF-ß1 secretion.

6. RGDS stimulates TGF-ß1 secretion through ILK activity
The role of ILK in RGDS effect was tested by inhibiting ILK activity. HMC were transfected with a kinase dead (KD) ILK form and treated with 50 µM RGDS for 16 h. This treatment abrogated the stimulation of TGF-ß1 secretion as well as the increased promoter activity induced by RGDS (Fig. 2 ), indicating that ILK is essential in the signal transduction of the RGDS effect on the regulation of TGF-ß1 synthesis.



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  		Figure 2. Inhibition of ILK by kinase dead (KD)-ILK transfection abrogates the stimulation of TGF-ß1 synthesis and promoter activity induced by RGDS. A) HMC transfected with KD for 16 h had reduced ILK activity (upper inset) and reduced TGF-ß1 secretion (ELISA) in response to 50 µM RGDS for 16 h. Cells transfected with the empty vector showed increased ILK activity and secretion of TGF-ß1 in response to RGDS. N = 6, *P < 0.05 vs. control. B) HMC cotransfected with KD and pGL3 TGF-ß1 promoter luciferase cDNA had reduced TGF-ß1 promoter activity, as determined by luciferase activity, vs. those cotransfected with the empty vector and pGL3 TGF-ß1 promoter luciferase cDNA in the presence of 50 µM RGDS for 16 h. RGES did not modify TGF-ß1 promoter activity. N = 5, *P < 0.05 vs. control (untreated cells).

CONCLUSIONS AND SIGNIFICANCE

The results presented here clearly demonstrate that RGD-containing peptides can play a role in regulating TGF-ß1 synthesis in cultured HMC. RGDS has the following actions: 1) it increases the activity of the TGF-ß1 mRNA promoter, 2) it augments the steady state levels of this mRNA, and 3) it induces the release of immunoreactive TGF-ß1 to the incubation media. Cell integrins interact with certain ECM proteins through RGD motifs, and our results support a role for extracellular matrix in the regulation of TGF-ß1 synthesis and secretion. Since this cytokine is one of the main modulators of ECM protein synthesis and has a pivotal role in the progression of organ damage, the present data support the role of RGD-dependent signals in the modulation of this damage.

It is generally accepted that RGD actions are linked to its interaction with integrin. However, alternative mechanisms have been proposed. For instance, RGD-containing peptides are able to enter directly into some tumor cells without integrin interactions and induce apoptosis by activation of caspases. For this reason, we evaluated the effect of RGDS on TGF-ß1 mRNA expression and secretion in the presence of blocking antibodies against different integrin subunits. Our results show that anti-integrin antibodies prevent RGDS-dependent TGF-ß1 stimulation. {alpha}V and ß1, the most widely studied integrins that interact with fibronectin and RGD motifs, are involved in this response.

RGDS increases the tyrosine phosphorylation of HMC proteins as well as ILK activity. In the case of tyrosine kinases, similar effects have been demonstrated in other cell types whereas the involvement of ILK, a serine-threonine kinase, in RGD effects has been studied less. To test the relevance of these two types of kinases in the stimulation of TGF-ß1 synthesis by RGDS, activities were blocked by pharmacological or transfection means. Neither herbimycin nor genistein modified TGF-ß1 secretion. These results do not support a role for integrin-linked tyrosine kinase pathways in the RGDS-induced TGF-ß1 secretion, but they do not exclude this possibility.

The role of the ILK pathway appears more evident. The carboxyl terminus of ILK interacts with the cytoplasmic domains of integrins ß1 and ß3. Transfection of HMC with KD, a dominant negative form of ILK where the glutamic acid 359 is mutated to lysine, blocked both the stimulation of ILK activity and TGF-ß1 secretion and promoter activity induced by RGDS. This suggests that RGDS-stimulated TGF-ß1 synthesis depends on ILK activation.

The mechanisms by which ILK modulates TGF-ß1 secretion were not explored in these experiments. This serine-threonine kinase triggers different intracellular pathways. For instance, ILK directly phosphorylates proteins such as PKB (serine 473), glycogen synthase kinase 3 (serine 9), or myosin light chain (serine 18), and some relationship could exist between these activated pathways and TGF-ß1 mRNA expression. The promoter of this cytokine has some AP-1-responsive elements, and ILK activates AP-1 through inhibition of GSK-3. Nevertheless, additional experiments are needed to clarify these mechanisms.

In summary, we demonstrated that RGDS up-regulates TGF-ß1 mRNA expression and TGF-ß1 secretion in cultured HMC. The activation pathway involves integrins {alpha}V and ß1 and the subsequent activation of ILK. Our data support a role for the ECM in the regulation of TGF-ß1 secretion, suggesting that qualitative changes in ECM composition may exert a regulatory influence on the release of profibrogenic cytokines by cells.



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  		Figure 3. Schematic diagram.

FOOTNOTES

1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0785fje; doi: 10.1096/fj.02-0785fje




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