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Mechanisms of osteopontin and CD44 as metastatic principles in prostate cancer cells.

Desai B, Rogers MJ, Chellaiah MA - Mol. Cancer (2007)

Bottom Line: BPs inhibits the mevalonate pathway, which in turn, prevents the prenylation of a number of small GTPases.Attenuation of Rho GTPase activation by BPs may have contributed to the down regulation of cell surface CD44/MMP-9 interaction, MMP-9 activation/secretion, and cell migration.The various steps involved in the above mentioned signaling pathway and/or the molecules regulating the activation of MMP-9 are potential therapeutic target.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedical Sciences, Dental School, University of Maryland, Baltimore, MD 21201, USA. bdesa002@umaryland.edu

ABSTRACT

Background: The expression level of osteopontin correlates with the metastatic potential of several tumors. Osteopontin is a well-characterized ligand for the alphavbeta3 integrin. The present study was undertaken to elucidate the possible role of osteopontin/alphavbeta3 signaling in prostate cancer cell migration.

Results: We generated stable prostate cancer cell (PC3) lines that over-express osteopontin (PC3/OPN), mutant OPN in the integrin binding-site (PC3/RGDDeltaRGA), and for OPN (PC3/SiRNA). The following observations were made in PC3/OPN cells as compared with PC3 cells: 1) an increase in multinucleated giant cells and RANKL expression; 2) an increase in CD44 surface expression, interaction of CD44/MMP-9 on the cell surface, MMP-9 activity in the conditioned medium, and cell migration; 3) western blot analysis of concentrated conditioned medium exhibited equal levels of MMP-9 protein in all PC3 cells. However, zymography analysis demonstrated that the levels of MMP-9 activity in the conditioned media reflect the CD44 surface expression pattern of the PC3 cell lines; 4) although MMP-9 and MMP-2 are secreted by PC3 cells, only the secretion of MMP-9 is regulated by OPN expression. A strong down regulation of the above-mentioned processes was observed in PC3/OPN (RGA) and PC3/SiRNA cells. PC3/OPN cells treated with bisphosphonate (BP) reproduce the down-regulation observed in PC3/OPN (RGA) and PC3/SiRNA cells.

Conclusion: Rho signaling plays a crucial role in CD44 surface expression. BPs inhibits the mevalonate pathway, which in turn, prevents the prenylation of a number of small GTPases. Attenuation of Rho GTPase activation by BPs may have contributed to the down regulation of cell surface CD44/MMP-9 interaction, MMP-9 activation/secretion, and cell migration. Taken together, these observations suggest that CD44 surface expression is an important event in the activation of MMP-9 and migration of prostate cancer cells. The various steps involved in the above mentioned signaling pathway and/or the molecules regulating the activation of MMP-9 are potential therapeutic target.

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Related in: MedlinePlus

Analysis of the effects of osteopontin (OPN) expression on MMP-9 activity. A. OPN expression levels were measured in different PC3 cell lines (indicated below each lane) by immunoblotting analysis. Untransfected PC3 cells (lane 1), PC3 cells transfected with pCEP4 vector (lane 2), pSilencer 4.1-CMV neo vector (lane 3), and Scrambled RNA construct (lane 10) were used as controls. Basal level expression of OPN was observed in these cells. An increase in OPN expression was observed in PC3 cells transfected with full length (PC3/OPN; lane 4) and mutated (PC3/OPN (RGA); lane 5) OPN cDNA. Individual clones (denoted as C1) that express maximum level of OPN is used for the studies shown here (lanes 4 and 5). PC3 cells transfected with four different SiRNA constructs exhibited expression of different levels of OPN (lanes 6–9). Immunoblotting with an antibody to GAPDH was used as a loading control (lower panel in A). B and C: Analysis of MMPs activity in the conditioned media of different PC3 cell lines by gelatin zymography: Several individual clones (10–15) were isolated from PC3 cells transfected with different OPN constructs (full length, mutated (RGDΔRGA), and SiRNA). Conditioned media collected from clones that express maximum (Figure B, lanes 3–10; C1–C4 in PC3/OPN and PC3/OPN(RGA)) and reduced levels (Figure C, lanes 3–7, C1–C5) of OPN protein were used for zymogram analysis Untransfected PC3 cells were used as control (lane1 in B and C). The activity of a recombinant MMP-9 protein containing pro- and active band was used as an identification marker (lane 1 in B and C). Gelatinolytic activities of both MMP-2 and MMP-9 are indicated by arrows (B and C). The results represent one of three separate experiments performed.
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Figure 1: Analysis of the effects of osteopontin (OPN) expression on MMP-9 activity. A. OPN expression levels were measured in different PC3 cell lines (indicated below each lane) by immunoblotting analysis. Untransfected PC3 cells (lane 1), PC3 cells transfected with pCEP4 vector (lane 2), pSilencer 4.1-CMV neo vector (lane 3), and Scrambled RNA construct (lane 10) were used as controls. Basal level expression of OPN was observed in these cells. An increase in OPN expression was observed in PC3 cells transfected with full length (PC3/OPN; lane 4) and mutated (PC3/OPN (RGA); lane 5) OPN cDNA. Individual clones (denoted as C1) that express maximum level of OPN is used for the studies shown here (lanes 4 and 5). PC3 cells transfected with four different SiRNA constructs exhibited expression of different levels of OPN (lanes 6–9). Immunoblotting with an antibody to GAPDH was used as a loading control (lower panel in A). B and C: Analysis of MMPs activity in the conditioned media of different PC3 cell lines by gelatin zymography: Several individual clones (10–15) were isolated from PC3 cells transfected with different OPN constructs (full length, mutated (RGDΔRGA), and SiRNA). Conditioned media collected from clones that express maximum (Figure B, lanes 3–10; C1–C4 in PC3/OPN and PC3/OPN(RGA)) and reduced levels (Figure C, lanes 3–7, C1–C5) of OPN protein were used for zymogram analysis Untransfected PC3 cells were used as control (lane1 in B and C). The activity of a recombinant MMP-9 protein containing pro- and active band was used as an identification marker (lane 1 in B and C). Gelatinolytic activities of both MMP-2 and MMP-9 are indicated by arrows (B and C). The results represent one of three separate experiments performed.

Mentions: To determine the OPN/αvβ3-mediated signaling mechanisms involved in prostate cancer cell migration, we generated different PC3 cell lines as described in the Methods section. OPN expression levels were detected by immunoblotting with an antibody to OPN (Figure 1A; top panel. Approximately 10–15 clones were analyzed for high expression of OPN by Western blotting with an antibody to OPN. Among the clones tested, we chose one that exhibited high expression (>10 times) of full length OPN (clone C1; lane 4) and mutant (RGDΔRGA) OPN (Clone C1; lane 5). Untransfected and pCEP-4 vector transfected PC3 cells (lanes 1 and 2) were used as controls. To reduce endogenous OPN in PC3 cells, we generated four different OPN SiRNA constructs in the pSilencer vector (lanes 6–9). Scrambled RNA constructs (lane 10) and vector (pSilencer 4.1-CMV neo vector) -transfected cells (lane 3) were used as controls. A significant decrease in OPN expression was observed in PC3 cells transfected with a SiRNA construct. This construct was targeted to human OPN cDNA sequence 383–403 (lane 7). Immunoblot that is shown in panel A was stripped and blotted with an antibody to GAPDH (Figure 1A, bottom panel) as a loading control.


Mechanisms of osteopontin and CD44 as metastatic principles in prostate cancer cells.

Desai B, Rogers MJ, Chellaiah MA - Mol. Cancer (2007)

Analysis of the effects of osteopontin (OPN) expression on MMP-9 activity. A. OPN expression levels were measured in different PC3 cell lines (indicated below each lane) by immunoblotting analysis. Untransfected PC3 cells (lane 1), PC3 cells transfected with pCEP4 vector (lane 2), pSilencer 4.1-CMV neo vector (lane 3), and Scrambled RNA construct (lane 10) were used as controls. Basal level expression of OPN was observed in these cells. An increase in OPN expression was observed in PC3 cells transfected with full length (PC3/OPN; lane 4) and mutated (PC3/OPN (RGA); lane 5) OPN cDNA. Individual clones (denoted as C1) that express maximum level of OPN is used for the studies shown here (lanes 4 and 5). PC3 cells transfected with four different SiRNA constructs exhibited expression of different levels of OPN (lanes 6–9). Immunoblotting with an antibody to GAPDH was used as a loading control (lower panel in A). B and C: Analysis of MMPs activity in the conditioned media of different PC3 cell lines by gelatin zymography: Several individual clones (10–15) were isolated from PC3 cells transfected with different OPN constructs (full length, mutated (RGDΔRGA), and SiRNA). Conditioned media collected from clones that express maximum (Figure B, lanes 3–10; C1–C4 in PC3/OPN and PC3/OPN(RGA)) and reduced levels (Figure C, lanes 3–7, C1–C5) of OPN protein were used for zymogram analysis Untransfected PC3 cells were used as control (lane1 in B and C). The activity of a recombinant MMP-9 protein containing pro- and active band was used as an identification marker (lane 1 in B and C). Gelatinolytic activities of both MMP-2 and MMP-9 are indicated by arrows (B and C). The results represent one of three separate experiments performed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1828067&req=5

Figure 1: Analysis of the effects of osteopontin (OPN) expression on MMP-9 activity. A. OPN expression levels were measured in different PC3 cell lines (indicated below each lane) by immunoblotting analysis. Untransfected PC3 cells (lane 1), PC3 cells transfected with pCEP4 vector (lane 2), pSilencer 4.1-CMV neo vector (lane 3), and Scrambled RNA construct (lane 10) were used as controls. Basal level expression of OPN was observed in these cells. An increase in OPN expression was observed in PC3 cells transfected with full length (PC3/OPN; lane 4) and mutated (PC3/OPN (RGA); lane 5) OPN cDNA. Individual clones (denoted as C1) that express maximum level of OPN is used for the studies shown here (lanes 4 and 5). PC3 cells transfected with four different SiRNA constructs exhibited expression of different levels of OPN (lanes 6–9). Immunoblotting with an antibody to GAPDH was used as a loading control (lower panel in A). B and C: Analysis of MMPs activity in the conditioned media of different PC3 cell lines by gelatin zymography: Several individual clones (10–15) were isolated from PC3 cells transfected with different OPN constructs (full length, mutated (RGDΔRGA), and SiRNA). Conditioned media collected from clones that express maximum (Figure B, lanes 3–10; C1–C4 in PC3/OPN and PC3/OPN(RGA)) and reduced levels (Figure C, lanes 3–7, C1–C5) of OPN protein were used for zymogram analysis Untransfected PC3 cells were used as control (lane1 in B and C). The activity of a recombinant MMP-9 protein containing pro- and active band was used as an identification marker (lane 1 in B and C). Gelatinolytic activities of both MMP-2 and MMP-9 are indicated by arrows (B and C). The results represent one of three separate experiments performed.
Mentions: To determine the OPN/αvβ3-mediated signaling mechanisms involved in prostate cancer cell migration, we generated different PC3 cell lines as described in the Methods section. OPN expression levels were detected by immunoblotting with an antibody to OPN (Figure 1A; top panel. Approximately 10–15 clones were analyzed for high expression of OPN by Western blotting with an antibody to OPN. Among the clones tested, we chose one that exhibited high expression (>10 times) of full length OPN (clone C1; lane 4) and mutant (RGDΔRGA) OPN (Clone C1; lane 5). Untransfected and pCEP-4 vector transfected PC3 cells (lanes 1 and 2) were used as controls. To reduce endogenous OPN in PC3 cells, we generated four different OPN SiRNA constructs in the pSilencer vector (lanes 6–9). Scrambled RNA constructs (lane 10) and vector (pSilencer 4.1-CMV neo vector) -transfected cells (lane 3) were used as controls. A significant decrease in OPN expression was observed in PC3 cells transfected with a SiRNA construct. This construct was targeted to human OPN cDNA sequence 383–403 (lane 7). Immunoblot that is shown in panel A was stripped and blotted with an antibody to GAPDH (Figure 1A, bottom panel) as a loading control.

Bottom Line: BPs inhibits the mevalonate pathway, which in turn, prevents the prenylation of a number of small GTPases.Attenuation of Rho GTPase activation by BPs may have contributed to the down regulation of cell surface CD44/MMP-9 interaction, MMP-9 activation/secretion, and cell migration.The various steps involved in the above mentioned signaling pathway and/or the molecules regulating the activation of MMP-9 are potential therapeutic target.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedical Sciences, Dental School, University of Maryland, Baltimore, MD 21201, USA. bdesa002@umaryland.edu

ABSTRACT

Background: The expression level of osteopontin correlates with the metastatic potential of several tumors. Osteopontin is a well-characterized ligand for the alphavbeta3 integrin. The present study was undertaken to elucidate the possible role of osteopontin/alphavbeta3 signaling in prostate cancer cell migration.

Results: We generated stable prostate cancer cell (PC3) lines that over-express osteopontin (PC3/OPN), mutant OPN in the integrin binding-site (PC3/RGDDeltaRGA), and for OPN (PC3/SiRNA). The following observations were made in PC3/OPN cells as compared with PC3 cells: 1) an increase in multinucleated giant cells and RANKL expression; 2) an increase in CD44 surface expression, interaction of CD44/MMP-9 on the cell surface, MMP-9 activity in the conditioned medium, and cell migration; 3) western blot analysis of concentrated conditioned medium exhibited equal levels of MMP-9 protein in all PC3 cells. However, zymography analysis demonstrated that the levels of MMP-9 activity in the conditioned media reflect the CD44 surface expression pattern of the PC3 cell lines; 4) although MMP-9 and MMP-2 are secreted by PC3 cells, only the secretion of MMP-9 is regulated by OPN expression. A strong down regulation of the above-mentioned processes was observed in PC3/OPN (RGA) and PC3/SiRNA cells. PC3/OPN cells treated with bisphosphonate (BP) reproduce the down-regulation observed in PC3/OPN (RGA) and PC3/SiRNA cells.

Conclusion: Rho signaling plays a crucial role in CD44 surface expression. BPs inhibits the mevalonate pathway, which in turn, prevents the prenylation of a number of small GTPases. Attenuation of Rho GTPase activation by BPs may have contributed to the down regulation of cell surface CD44/MMP-9 interaction, MMP-9 activation/secretion, and cell migration. Taken together, these observations suggest that CD44 surface expression is an important event in the activation of MMP-9 and migration of prostate cancer cells. The various steps involved in the above mentioned signaling pathway and/or the molecules regulating the activation of MMP-9 are potential therapeutic target.

Show MeSH
Related in: MedlinePlus