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Interfering with TGFbeta-induced Smad3 nuclear accumulation differentially affects TGFbeta-dependent gene expression.

Lindemann RK, Nordheim A, Dittmer J - Mol. Cancer (2003)

Bottom Line: Our results show that these inhibitors delay the onset of TGFbeta-induced nuclear accumulation of Smad3 and reduces its amplitude.This effect was accompanied by a strong reduction in TGFbeta-responsivess of the slow-responder genes pthrp, pai-1 and upa, while the reactivity of the fast-responder gene smad7 to TGFbeta remained almost unchanged.Neither was the TGFbeta response of the fast-responder ese-1/esx gene, whose expression we found to be strongly downregulated by TGFbeta, affected by the inhibitors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Zellbiologie, Abteilung Molekularbiologie, Universität Tübingen, Auf der Morgenstelle 15, 72076 Tübingen, Germany. ralph.lindemann@uni-tuebingen.de

ABSTRACT

Background: Transforming growth factor-beta (TGFbeta) plays an important role in late-stage carcinogenesis by stimulating invasive behavior of cancer cells, promoting neo-angiogenesis and by helping cancer cells to escape surveillance by the immune system. It also supports colonization of the bone by metastatic breast cancer cells by increasing expression of osteolytic parathyroid hormone-related protein (PTHrP). Interfering with TGFbeta signalling may thus weaken the malignant properties of cancer cells. We investigated to what extent two inhibitors, SB-202190 and SB-203580, interfere with TGFbeta-signalling in invasive MDA-MB-231 breast cancer cells. These compounds, formerly used as p38-MAPK-specific inhibitors, were recently also demonstrated to inhibit TGFbeta type I receptor kinase.

Results: Our results show that these inhibitors delay the onset of TGFbeta-induced nuclear accumulation of Smad3 and reduces its amplitude. This effect was accompanied by a strong reduction in TGFbeta-responsivess of the slow-responder genes pthrp, pai-1 and upa, while the reactivity of the fast-responder gene smad7 to TGFbeta remained almost unchanged. Neither was the TGFbeta response of the fast-responder ese-1/esx gene, whose expression we found to be strongly downregulated by TGFbeta, affected by the inhibitors.

Conclusion: The data show that SB-202190 and SB-203580 suppress TGFbeta-dependent activation of genes that are important for the acquisition of invasive behavior, while having no effect on the expression of the natural TGFbeta inhibitor Smad7. This suggests that these compounds are potent inhibitors of malignant behavior of cancer cells.

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SB inhibitors deregulate TGFβ induced Smad3 nuclear accumulation. A, Western Blotting analysis of whole cell and nuclear extracts of cells treated with 5 ng/ml TGFβ for 30 min after 45 min preincubation with DMSO or SB-202190 (10 μM) and SB-203580 (20 μM). Smad3 protein was detected using a mouse monoclonal Smad3 antibody from Santa Cruz. B, Western Blotting analysis as in A using nuclear extracts of cells stimulated with TGFβ for the indicated times after 45 min preincubation with DMSO or SB-203580 (20 μM). C, same as in A with inhibitors SB-203580 (20 μM), PD98059 (40 μM), Y-27632 (20 μM) or PMA (25 nM), respectively. Coomassie staining and reprobing of blots with a control antibody (PKCα) ensured equal protein loading for all lanes (not shown).
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Figure 2: SB inhibitors deregulate TGFβ induced Smad3 nuclear accumulation. A, Western Blotting analysis of whole cell and nuclear extracts of cells treated with 5 ng/ml TGFβ for 30 min after 45 min preincubation with DMSO or SB-202190 (10 μM) and SB-203580 (20 μM). Smad3 protein was detected using a mouse monoclonal Smad3 antibody from Santa Cruz. B, Western Blotting analysis as in A using nuclear extracts of cells stimulated with TGFβ for the indicated times after 45 min preincubation with DMSO or SB-203580 (20 μM). C, same as in A with inhibitors SB-203580 (20 μM), PD98059 (40 μM), Y-27632 (20 μM) or PMA (25 nM), respectively. Coomassie staining and reprobing of blots with a control antibody (PKCα) ensured equal protein loading for all lanes (not shown).

Mentions: It has recently been shown that SB-202190 and SB-203580 are able to inhibit TGFβ-induced phosphorylation of Smad3 by the TGFβ-receptor I kinase (ALK5) and to interfere with nuclear localization of Smad3 [17]. Nuclear translocation of Smad3 results directly from TGFβ-induced Smad3 phosphorylation and is, therefore, a measure of the activation status of ALK5. We wanted to know to what extent SB-202190 and SB-203580 interfere with TGFβ-dependent Smad3 nuclear import in the invasive MDA-MB-231 breast cancer cell line. We prepared nuclear and whole cell extracts from cells treated with TGFβ with or without preincubation of one of the SB inhibitors and analyzed those extracts for reactivity to a Smad2,3-specific antibody by Western blot analysis. This antibody primarily interacts with Smad3, but, to some extent, it is also able to recognize Smad2. Based on Northern blot hybridization data, Smad3, but not Smad2, is expressed in MDA-MB-231 cells [21]. Hence, the data presented in Fig. 2 show the abundance of Smad3 in the nuclear and in the whole cell extracts. We found that preincubation of cells with either SB-202190 or SB-203580 decreased the level of nuclear Smad3 in the presence of TGFβ (Fig. 2A), whereas the total Smad3 protein level, as assayed in whole cell extracts, was not altered.


Interfering with TGFbeta-induced Smad3 nuclear accumulation differentially affects TGFbeta-dependent gene expression.

Lindemann RK, Nordheim A, Dittmer J - Mol. Cancer (2003)

SB inhibitors deregulate TGFβ induced Smad3 nuclear accumulation. A, Western Blotting analysis of whole cell and nuclear extracts of cells treated with 5 ng/ml TGFβ for 30 min after 45 min preincubation with DMSO or SB-202190 (10 μM) and SB-203580 (20 μM). Smad3 protein was detected using a mouse monoclonal Smad3 antibody from Santa Cruz. B, Western Blotting analysis as in A using nuclear extracts of cells stimulated with TGFβ for the indicated times after 45 min preincubation with DMSO or SB-203580 (20 μM). C, same as in A with inhibitors SB-203580 (20 μM), PD98059 (40 μM), Y-27632 (20 μM) or PMA (25 nM), respectively. Coomassie staining and reprobing of blots with a control antibody (PKCα) ensured equal protein loading for all lanes (not shown).
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Figure 2: SB inhibitors deregulate TGFβ induced Smad3 nuclear accumulation. A, Western Blotting analysis of whole cell and nuclear extracts of cells treated with 5 ng/ml TGFβ for 30 min after 45 min preincubation with DMSO or SB-202190 (10 μM) and SB-203580 (20 μM). Smad3 protein was detected using a mouse monoclonal Smad3 antibody from Santa Cruz. B, Western Blotting analysis as in A using nuclear extracts of cells stimulated with TGFβ for the indicated times after 45 min preincubation with DMSO or SB-203580 (20 μM). C, same as in A with inhibitors SB-203580 (20 μM), PD98059 (40 μM), Y-27632 (20 μM) or PMA (25 nM), respectively. Coomassie staining and reprobing of blots with a control antibody (PKCα) ensured equal protein loading for all lanes (not shown).
Mentions: It has recently been shown that SB-202190 and SB-203580 are able to inhibit TGFβ-induced phosphorylation of Smad3 by the TGFβ-receptor I kinase (ALK5) and to interfere with nuclear localization of Smad3 [17]. Nuclear translocation of Smad3 results directly from TGFβ-induced Smad3 phosphorylation and is, therefore, a measure of the activation status of ALK5. We wanted to know to what extent SB-202190 and SB-203580 interfere with TGFβ-dependent Smad3 nuclear import in the invasive MDA-MB-231 breast cancer cell line. We prepared nuclear and whole cell extracts from cells treated with TGFβ with or without preincubation of one of the SB inhibitors and analyzed those extracts for reactivity to a Smad2,3-specific antibody by Western blot analysis. This antibody primarily interacts with Smad3, but, to some extent, it is also able to recognize Smad2. Based on Northern blot hybridization data, Smad3, but not Smad2, is expressed in MDA-MB-231 cells [21]. Hence, the data presented in Fig. 2 show the abundance of Smad3 in the nuclear and in the whole cell extracts. We found that preincubation of cells with either SB-202190 or SB-203580 decreased the level of nuclear Smad3 in the presence of TGFβ (Fig. 2A), whereas the total Smad3 protein level, as assayed in whole cell extracts, was not altered.

Bottom Line: Our results show that these inhibitors delay the onset of TGFbeta-induced nuclear accumulation of Smad3 and reduces its amplitude.This effect was accompanied by a strong reduction in TGFbeta-responsivess of the slow-responder genes pthrp, pai-1 and upa, while the reactivity of the fast-responder gene smad7 to TGFbeta remained almost unchanged.Neither was the TGFbeta response of the fast-responder ese-1/esx gene, whose expression we found to be strongly downregulated by TGFbeta, affected by the inhibitors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Zellbiologie, Abteilung Molekularbiologie, Universität Tübingen, Auf der Morgenstelle 15, 72076 Tübingen, Germany. ralph.lindemann@uni-tuebingen.de

ABSTRACT

Background: Transforming growth factor-beta (TGFbeta) plays an important role in late-stage carcinogenesis by stimulating invasive behavior of cancer cells, promoting neo-angiogenesis and by helping cancer cells to escape surveillance by the immune system. It also supports colonization of the bone by metastatic breast cancer cells by increasing expression of osteolytic parathyroid hormone-related protein (PTHrP). Interfering with TGFbeta signalling may thus weaken the malignant properties of cancer cells. We investigated to what extent two inhibitors, SB-202190 and SB-203580, interfere with TGFbeta-signalling in invasive MDA-MB-231 breast cancer cells. These compounds, formerly used as p38-MAPK-specific inhibitors, were recently also demonstrated to inhibit TGFbeta type I receptor kinase.

Results: Our results show that these inhibitors delay the onset of TGFbeta-induced nuclear accumulation of Smad3 and reduces its amplitude. This effect was accompanied by a strong reduction in TGFbeta-responsivess of the slow-responder genes pthrp, pai-1 and upa, while the reactivity of the fast-responder gene smad7 to TGFbeta remained almost unchanged. Neither was the TGFbeta response of the fast-responder ese-1/esx gene, whose expression we found to be strongly downregulated by TGFbeta, affected by the inhibitors.

Conclusion: The data show that SB-202190 and SB-203580 suppress TGFbeta-dependent activation of genes that are important for the acquisition of invasive behavior, while having no effect on the expression of the natural TGFbeta inhibitor Smad7. This suggests that these compounds are potent inhibitors of malignant behavior of cancer cells.

Show MeSH
Related in: MedlinePlus