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Endothelin signaling axis activates osteopontin expression through PI3 kinase pathway in A375 melanoma cells
Journal of Dermatological Science (2008) 52, 130—148
www.intl.elsevierhealth.com/journals/jods
LETTER TO THE EDITOR Endothelin signaling axis activates osteopontin expression through PI3 kinase pathway in A375 melanoma cells There is an abnormal transcriptional activation of the endothelin 3 (ET-3) and endothelin receptor B (ETRB) in a high percentage of metastatic melanoma. This is accompanied by altered expression of extracellular matrix protein, such as osteopontin (OPN) [1]. We investigated if the increased expression of OPN in melanoma cells is controlled by activation of endothelin pathway. Packer et al, found that down-regulated PTEN caused activation of phosphatidylinositol 3 (PI3) kinase and then the OPN up-regulation [2], thus confirming the downstream status of OPN to the PI3 kinase pathway. Therefore, we further investigate if the increased expression of OPN in melanoma cells also involves signaling molecule PI3 kinase. To verify our postulation we detected the effects of ET-3 on the expression of OPN in cultured A375 cells by RT-PCR and Western blot. The methods carried out in our experiment were as described by Kim [3]. Basically, A375 cells (from ATCC) were cultured and then seeded to six-well plates at a density of 2.5 105/well, and allowed to recover for 24 h. To activate the ETRB pathway, fresh medium containing various concentrations (0, 1 nM, 10 nM and 100 nM) of ET-3 (Alexis Corporation, Lausen, Switzerland) was added to the wells after suction removal of existing medium and allowed to incubate by 24 h. The cells were then harvested for analysis of OPN expression. To examine the time course of OPN response to ET-3 stimulation, the A375 cells were treated with 100 nM ET-3 for 0, 6, 12, 24, 30 and 36 h before harvesting for OPN assay. To examine the effect of ET-3 antagonist on OPN expression, the A375 cells were treated with BQ788 peptide (Alexis
§ China National Natural Science Foundation (to Huang Changzheng, grant no. 30671891) and the Young Scientists Fund Program of National Science Fund Program from National Natural Science Foundation of China (to Tao Juan, grant no. 30500437).
Corporation, Lausen, Switzerland) at 1 mM for 1 h, and then changed into medium containing both BQ788 (1 mM) and ET-3 (100 nM) and allowed to grow for 24 h before harvesting and OPN assay. To examine the effect of PI3 kinase pathway on OPN expression, the A375 cells were pre-treated with LY294002 (Cell Signaling Technology Inc., MA, USA) at 20 mM or 50 mM for one hour before addition of ET-3 at 100 nM and incubated for 24 h before harvesting. Each treatment condition was performed in triplicate. The harvested cells were then used for OPN assay. The photographs of RT-PCR gel electrophoresis and Western blot films were scanned with Bio-Rad 1D gel quantification Quantity One system. SPSS 11.5 was used to analyze the statistical significance of the experimental data. The data were expressed as average of three triplicates plus standard deviation. Student’s t-test was used to calculate the significance with P < 0.05 as significant. As shown in Fig. 1A and D strong enhancement of OPN mRNA expression was observed under ET-3 treatment in a concentration-dependent manner. The difference was associated with strong increase of OPN protein (Fig. 2G and J). At 100 nM ET-3 treatment, A375 cells produce almost fivefold more OPN protein compared with non-treatment controls in the absence of ET-3 (P < 0.05, t-test, Fig. 2J). Since the strongest effect of ET-3 on OPN expression was at 100 nM, we then used this concentration to determine the time course of OPN response. In terms of OPN mRNA expression, the effect peaked at 24 h before declining (Fig. 1B and E). Similarly, the OPN protein response also peaks at 24 h (Fig. 2H and K). As shown in Figs. 1C, 1F and 2I, 2L, the ET-3 induced increase of OPN mRNA and protein was significantly blocked by the BQ788 peptide inhibitor by at least 70% (P < 0.05). The expression of OPN under the stimulation of ET-3 was also blocked by a specific inhibitor of PI3 kinase pathway, LY294002, in a concentration-dependent manner (P < 0.05). The decreased effect of OPN by BQ788 was more obvious than by LY294002. Melanoma cells have been observed to aberrantly express a wide array of genes, including OPN and
0923-1811/$30.00 . Crown Copyright # 2008 Published by Elsevier Ireland Ltd on behalf of Japanese Society of Investigative Dermatology. All rights reserved.
Letters to the Editor
131
Fig. 1 OPN mRNA expression: in different concentration of exogenous ET3 (A and D), temporal course (B and E), inhibitory effects of BQ788 and LY294002 (C and F). (A—F) RT-PCR results, for control beta-actin was used. (A) Induction of osteopontin transcription by exogenous ET-3 at various concentrations. Lanes 1—4: 0, 10 nM, 50 nM and 100 nM ET-3 added to the growth medium. (B) Temporal course of ET-3 induction of osteopontin transcription. The cells were harvested at 0, 6, 12, 18, 24, 30 and 36 h (Lanes 1—7) after the addition of 100 nM ET-3. (C) Effect of BQ788 and LY294002 on ET-3 induced OPN transcription. Cultured A375 Cells were pre-treated with growth medium supplemented with no inhibitor (Lane 1), 1 mM BQ788 (Lane 2), 20 mM LY294002 (Lane 3) or 50 mM LY294002 (Lane 4) for 1 h. (D—F) The corresponding results of quantitative analysis of band intensities in (A—C), respectively.
ETRB gene [1]. However the relationship between these expression changes has not been studied previously. Using cultured melanoma cells A375 cells, which naturally produced significant levels of OPN protein, we generated evidence that activation of ETRB by ET-3 stimulation lead to significant enhancement of OPN expression, both at the mes-
senger RNA levels and in protein levels. Previous work by Lahav et al. demonstrated that activation of the ETRB was involved in melanoma cell survival [4]. The results presented here suggest that OPN is a down stream effecter molecule through which the ETRB pathway exerts its pro-invasion and prometastasis function.
Fig. 2 OPN protein expression: in different concentration of exogenous ET3 (G and J), temporal course (H and K), inhibitory effects of BQ788 and LY294002 (I and L). (G—L) Western blot results, for control GAPDH were used. (G—I) The corresponding Western blot results for Fig. 1(A—C), respectively. (J—L) The corresponding results of quantitative analysis of band intensities in (G—I), respectively.
132 In addition, we uncovered evidence that the endothelin-induced OPN expression is mediated at least in part by the PI3 kinase pathway since a specific inhibitor of this pathway can significantly down-regulate ET-3 induced enhancement of OPN expression. Functionally, OPN expression leads to increased cell migration, and increased survival of the melanoma cells in culture [1]. Therefore, blocking its expression, or inhibiting its function potentially has therapeutic value. However, OPN is also widely present in the bone and cartilage, and is up-regulated in soft tissues and joints in response to wound healing and tissue repair. Therefore, direct targeting of this protein may not offer enough specificity to reduce potential toxicity. Therefore, targeting the upstream regulators may offer an additional level at which cell specificity can be achieved. Our results suggest that indirectly targeting the upstream genes, such as the PI3 kinase or the ET-3 axis may serve as alternative mechanism of achieving OPN inhibition. Interestingly, the inhibitor of PI3 kinase just partially blocks the ET-3/ETRB axis’s stimulating effect on the expression of the OPN. It seems reasonable that ET-3/ETRB axis up-regulating the expression of OPN may also be mediated by other signalling pathways other than PI3K pathway. It has been documented that FAK (focal adhesion kinase) and MAPK (mitogen-activated protein kinase) pathway played an important role in the up-regulation of Ncadherin, a2b1 integrin, avb3 integrin, MMP-2 and MMP-9 induced by ET-3/ETRB axis [5]. Together with our results, we postulate that FAK and MAPK may be an applausive candidate.
References [1] Zhou Y, Dai DL, Martinka M, et al. Osteopontin expression correlates with melanoma invasion. J Invest Dermatol 2005;124:1044—52. [2] Packer L, Pavey S, Parker A, et al. Osteopontin is a downstream effector of the PI3-kinase pathway in melanomas that is inversely correlated with functional PTEN. Carcinogenesis 2006;27:1778—86. [3] Kim MS, Park MJ, Moon EJ, et al. Hyaluronic acid induces osteopontin via the phosphatidylinositol 3-kinase/Akt pathway to enhance the motility of human glioma cells. Cancer Res 2005;65:686—91. [4] Lahav R, Suva ML, Rimoldi D, Patterson PH, Stamenkovic I. Endothelin receptor B inhibition triggers apoptosis and enhances angiogenesis in melanomas. Cancer Res 2004;64: 8945—53. [5] Bagnato A, Rosano L, Spinella F, Di Castro V, Tecce R, Natali PG. Endothelin B receptor blockade inhibits dynamics of cell interactions and communications in melanoma cell progression. Cancer Res 2004;64:1436—43.
Letters to the Editor Huang Chang-zheng1 Department of Dermatology, Union Hospital, Wuhan, China Tian Jin1 Department of Infectious diseases, Union Hospital, Wuhan, China Tao Juan1 Liu Ye-qiang Li Yan Yang Ling-yun Zhang Jing Li Yan-qiu Chen Si-yuan Lin Neng-xing Department of Dermatology, Union Hospital, Wuhan, China Shen Guan-xin Faculty of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Tu Ya-ting* Department of Dermatology, Union Hospital, Wuhan, China Youwen Zhoua,b,** Institute of Dermatology, Anhui Medical University, Hefei, Anhui, China Department of Dermatology and Skin Science, University of British Columbia, Vancouver, British Columbia, Canada *Corresponding author at: Department of Dermatology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430022, PR China **Corresponding author at: Department of Dermatology and Skin Science, University of British Columbia, Vancouver, British Columbia, Canada E-mail addresses: [email protected] [email protected] 1
These authors contribute equally to this work. 13 December 2007
doi:10.1016/j.jdermsci.2008.05.006
www.intl.elsevierhealth.com/journals/jods
LETTER TO THE EDITOR Endothelin signaling axis activates osteopontin expression through PI3 kinase pathway in A375 melanoma cells There is an abnormal transcriptional activation of the endothelin 3 (ET-3) and endothelin receptor B (ETRB) in a high percentage of metastatic melanoma. This is accompanied by altered expression of extracellular matrix protein, such as osteopontin (OPN) [1]. We investigated if the increased expression of OPN in melanoma cells is controlled by activation of endothelin pathway. Packer et al, found that down-regulated PTEN caused activation of phosphatidylinositol 3 (PI3) kinase and then the OPN up-regulation [2], thus confirming the downstream status of OPN to the PI3 kinase pathway. Therefore, we further investigate if the increased expression of OPN in melanoma cells also involves signaling molecule PI3 kinase. To verify our postulation we detected the effects of ET-3 on the expression of OPN in cultured A375 cells by RT-PCR and Western blot. The methods carried out in our experiment were as described by Kim [3]. Basically, A375 cells (from ATCC) were cultured and then seeded to six-well plates at a density of 2.5 105/well, and allowed to recover for 24 h. To activate the ETRB pathway, fresh medium containing various concentrations (0, 1 nM, 10 nM and 100 nM) of ET-3 (Alexis Corporation, Lausen, Switzerland) was added to the wells after suction removal of existing medium and allowed to incubate by 24 h. The cells were then harvested for analysis of OPN expression. To examine the time course of OPN response to ET-3 stimulation, the A375 cells were treated with 100 nM ET-3 for 0, 6, 12, 24, 30 and 36 h before harvesting for OPN assay. To examine the effect of ET-3 antagonist on OPN expression, the A375 cells were treated with BQ788 peptide (Alexis
§ China National Natural Science Foundation (to Huang Changzheng, grant no. 30671891) and the Young Scientists Fund Program of National Science Fund Program from National Natural Science Foundation of China (to Tao Juan, grant no. 30500437).
Corporation, Lausen, Switzerland) at 1 mM for 1 h, and then changed into medium containing both BQ788 (1 mM) and ET-3 (100 nM) and allowed to grow for 24 h before harvesting and OPN assay. To examine the effect of PI3 kinase pathway on OPN expression, the A375 cells were pre-treated with LY294002 (Cell Signaling Technology Inc., MA, USA) at 20 mM or 50 mM for one hour before addition of ET-3 at 100 nM and incubated for 24 h before harvesting. Each treatment condition was performed in triplicate. The harvested cells were then used for OPN assay. The photographs of RT-PCR gel electrophoresis and Western blot films were scanned with Bio-Rad 1D gel quantification Quantity One system. SPSS 11.5 was used to analyze the statistical significance of the experimental data. The data were expressed as average of three triplicates plus standard deviation. Student’s t-test was used to calculate the significance with P < 0.05 as significant. As shown in Fig. 1A and D strong enhancement of OPN mRNA expression was observed under ET-3 treatment in a concentration-dependent manner. The difference was associated with strong increase of OPN protein (Fig. 2G and J). At 100 nM ET-3 treatment, A375 cells produce almost fivefold more OPN protein compared with non-treatment controls in the absence of ET-3 (P < 0.05, t-test, Fig. 2J). Since the strongest effect of ET-3 on OPN expression was at 100 nM, we then used this concentration to determine the time course of OPN response. In terms of OPN mRNA expression, the effect peaked at 24 h before declining (Fig. 1B and E). Similarly, the OPN protein response also peaks at 24 h (Fig. 2H and K). As shown in Figs. 1C, 1F and 2I, 2L, the ET-3 induced increase of OPN mRNA and protein was significantly blocked by the BQ788 peptide inhibitor by at least 70% (P < 0.05). The expression of OPN under the stimulation of ET-3 was also blocked by a specific inhibitor of PI3 kinase pathway, LY294002, in a concentration-dependent manner (P < 0.05). The decreased effect of OPN by BQ788 was more obvious than by LY294002. Melanoma cells have been observed to aberrantly express a wide array of genes, including OPN and
0923-1811/$30.00 . Crown Copyright # 2008 Published by Elsevier Ireland Ltd on behalf of Japanese Society of Investigative Dermatology. All rights reserved.
Letters to the Editor
131
Fig. 1 OPN mRNA expression: in different concentration of exogenous ET3 (A and D), temporal course (B and E), inhibitory effects of BQ788 and LY294002 (C and F). (A—F) RT-PCR results, for control beta-actin was used. (A) Induction of osteopontin transcription by exogenous ET-3 at various concentrations. Lanes 1—4: 0, 10 nM, 50 nM and 100 nM ET-3 added to the growth medium. (B) Temporal course of ET-3 induction of osteopontin transcription. The cells were harvested at 0, 6, 12, 18, 24, 30 and 36 h (Lanes 1—7) after the addition of 100 nM ET-3. (C) Effect of BQ788 and LY294002 on ET-3 induced OPN transcription. Cultured A375 Cells were pre-treated with growth medium supplemented with no inhibitor (Lane 1), 1 mM BQ788 (Lane 2), 20 mM LY294002 (Lane 3) or 50 mM LY294002 (Lane 4) for 1 h. (D—F) The corresponding results of quantitative analysis of band intensities in (A—C), respectively.
ETRB gene [1]. However the relationship between these expression changes has not been studied previously. Using cultured melanoma cells A375 cells, which naturally produced significant levels of OPN protein, we generated evidence that activation of ETRB by ET-3 stimulation lead to significant enhancement of OPN expression, both at the mes-
senger RNA levels and in protein levels. Previous work by Lahav et al. demonstrated that activation of the ETRB was involved in melanoma cell survival [4]. The results presented here suggest that OPN is a down stream effecter molecule through which the ETRB pathway exerts its pro-invasion and prometastasis function.
Fig. 2 OPN protein expression: in different concentration of exogenous ET3 (G and J), temporal course (H and K), inhibitory effects of BQ788 and LY294002 (I and L). (G—L) Western blot results, for control GAPDH were used. (G—I) The corresponding Western blot results for Fig. 1(A—C), respectively. (J—L) The corresponding results of quantitative analysis of band intensities in (G—I), respectively.
132 In addition, we uncovered evidence that the endothelin-induced OPN expression is mediated at least in part by the PI3 kinase pathway since a specific inhibitor of this pathway can significantly down-regulate ET-3 induced enhancement of OPN expression. Functionally, OPN expression leads to increased cell migration, and increased survival of the melanoma cells in culture [1]. Therefore, blocking its expression, or inhibiting its function potentially has therapeutic value. However, OPN is also widely present in the bone and cartilage, and is up-regulated in soft tissues and joints in response to wound healing and tissue repair. Therefore, direct targeting of this protein may not offer enough specificity to reduce potential toxicity. Therefore, targeting the upstream regulators may offer an additional level at which cell specificity can be achieved. Our results suggest that indirectly targeting the upstream genes, such as the PI3 kinase or the ET-3 axis may serve as alternative mechanism of achieving OPN inhibition. Interestingly, the inhibitor of PI3 kinase just partially blocks the ET-3/ETRB axis’s stimulating effect on the expression of the OPN. It seems reasonable that ET-3/ETRB axis up-regulating the expression of OPN may also be mediated by other signalling pathways other than PI3K pathway. It has been documented that FAK (focal adhesion kinase) and MAPK (mitogen-activated protein kinase) pathway played an important role in the up-regulation of Ncadherin, a2b1 integrin, avb3 integrin, MMP-2 and MMP-9 induced by ET-3/ETRB axis [5]. Together with our results, we postulate that FAK and MAPK may be an applausive candidate.
References [1] Zhou Y, Dai DL, Martinka M, et al. Osteopontin expression correlates with melanoma invasion. J Invest Dermatol 2005;124:1044—52. [2] Packer L, Pavey S, Parker A, et al. Osteopontin is a downstream effector of the PI3-kinase pathway in melanomas that is inversely correlated with functional PTEN. Carcinogenesis 2006;27:1778—86. [3] Kim MS, Park MJ, Moon EJ, et al. Hyaluronic acid induces osteopontin via the phosphatidylinositol 3-kinase/Akt pathway to enhance the motility of human glioma cells. Cancer Res 2005;65:686—91. [4] Lahav R, Suva ML, Rimoldi D, Patterson PH, Stamenkovic I. Endothelin receptor B inhibition triggers apoptosis and enhances angiogenesis in melanomas. Cancer Res 2004;64: 8945—53. [5] Bagnato A, Rosano L, Spinella F, Di Castro V, Tecce R, Natali PG. Endothelin B receptor blockade inhibits dynamics of cell interactions and communications in melanoma cell progression. Cancer Res 2004;64:1436—43.
Letters to the Editor Huang Chang-zheng1 Department of Dermatology, Union Hospital, Wuhan, China Tian Jin1 Department of Infectious diseases, Union Hospital, Wuhan, China Tao Juan1 Liu Ye-qiang Li Yan Yang Ling-yun Zhang Jing Li Yan-qiu Chen Si-yuan Lin Neng-xing Department of Dermatology, Union Hospital, Wuhan, China Shen Guan-xin Faculty of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Tu Ya-ting* Department of Dermatology, Union Hospital, Wuhan, China Youwen Zhoua,b,** Institute of Dermatology, Anhui Medical University, Hefei, Anhui, China Department of Dermatology and Skin Science, University of British Columbia, Vancouver, British Columbia, Canada *Corresponding author at: Department of Dermatology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430022, PR China **Corresponding author at: Department of Dermatology and Skin Science, University of British Columbia, Vancouver, British Columbia, Canada E-mail addresses: [email protected] [email protected] 1
These authors contribute equally to this work. 13 December 2007
doi:10.1016/j.jdermsci.2008.05.006