Identification of Benzo[a]pyrene-InducibleCis-Acting Elements Within c-Ha-rasTranscriptional Regulatory Sequences

Abstract

Previous studies in this laboratory have demonstrated that transcriptional deregulation of c-Ha-ras expression is associated with the induction and maintenance of proliferative vascular smooth muscle cell (SMC) phenotypes by benzo[a]pyrene (BaP). We examined previously undescribed cis-acting elements within the proximal 5' regulatory region of c-Ha-ras (-550 to +220) for their ability to influence BaP-induced transcription in murine SMCs. BaP-inducible DNA binding activity was demonstrated at a site located -30 relative to the major start site cluster at +1 that exhibits extensive homology to a consensus aryl hydrocarbon response element (AHRE), as well as a site located at -543 that contains a consensus electrophile response element (EpRE). In vitro cross-linking studies revealed the specific interaction of 104- and 96-kDa proteins with the putative AHRE and of an 80-kDa protein with the EpRE. The use of monoclonal antibodies to the aryl hydrocarbon receptor transcription factor in competition electrophoretic mobility shift assays indicated this protein is specifically induced by BaP to interact at the AHRE within the c-Ha-ras 5' regulatory region. Transient transfection with an Ha-ras promoter construct containing the putative AHRE but lacking the EpRE linked to the chloramphenicol acetyl transferase reporter gene, followed by challenge with BaP (0.3, 3.0, and 30 microM), revealed transcriptional activation that was not statistically significant. However, insertion of an oligonucleotide composed of the EpRE immediately upstream of basal sequences at -330 was associated with strong activation of transcription by BaP. These data indicate that c-Ha-ras gene expression is modulated by BaP via a complex mechanism that likely involves interactions among multiple regulatory elements. We conclude that c-Ha-ras expression is regulated by BaP at the transcriptional level, a response that may constitute an epigenetic basis of atherogenesis.

Identification of Albumin Precursor Protein, Phi AP3, and α-Smooth Muscle Actin as Novel Components of Redox Sensing Machinery in Vascular Smooth Muscle Cells

“…2A). Two prominent bands of 80 and 100 kDa were present in fraction 6, a finding consistent with previous UV cross-linking studies identifying an 80-kDa protein as an ARE-interacting protein (Bral and Ramos, 1997). A 42-kDa protein was also predominant in this fraction.…”

Identification of Albumin Precursor Protein, Phi AP3, and α-Smooth Muscle Actin as Novel Components of Redox Sensing Machinery in Vascular Smooth Muscle Cells

“…2A). Two prominent bands of 80 and 100 kDa were present in fraction 6, a finding consistent with previous UV cross-linking studies identifying an 80-kDa protein as an ARE-interacting protein (Bral and Ramos, 1997). A 42-kDa protein was also predominant in this fraction.…”

Activation profiles of HSPA5 during the glomerular mesangial cell stress response to chemical injury

“…Concentrations of 1 and 10 M HgCl 2 and Pb 2ϩ acetate are within the range that induces ER stress in rat C6 glioma cells (Qian et al 1995(Qian et al , 1999(Qian et al , 2001(Qian et al , 2005a, and consistent with environmental concentrations of these metals (Florea and Busselberg, 2006). Likewise, BaP exposures at 3 M span the range of estimated human exposures 0.171-1.64 g/day (Busbee et al 1990;USEPA 1990;Bral and Ramos 1997). None of the treatment regimens tested, except for 30 M BaP, affect cell viability (data not shown).…”

“…Severe functional deficits, including enzymuria, proteinuria, impaired transport, and depressed glomerular filtration rate, occur at exposures Ͼ50 g/dL (Lilis et al 1968;Baker et al 1980;Hong et al 1980;ATSDR, 2005). As described for the heavy metals, BaP exposures at 3 M span the range of estimated human exposures 0.171-1.64 g/d (Busbee et al 1990;USEPA 1990;Bral and Ramos 1997).…”

Altered Gene Expression Patterns in MCF-7 Cells Induced by the Urban Dust Particulate Complex Mixture Standard Reference Material 1649a

“…PAHs can induce tumor promotion through several, likely combinatorial, mechanisms including negative effects on transcription of tumor-suppressor genes (42), transcriptional activation of proto-oncogenes (43), and activation of enzymes that oxidize procarcinogenic PAHs to reactive metabolites (44). To date, no mutations in the BRCA-1 gene have been identified in sporadic breast cancers, whereas the expression levels of BRCA-1 in breast tumors are lower than those observed in normal mammary tissue (45).…”

Identification of Albumin Precursor Protein, Phi AP3, and α-Smooth Muscle Actin as Novel Components of Redox Sensing Machinery in Vascular Smooth Muscle Cells

“…2A). Two prominent bands of 80 and 100 kDa were present in fraction 6, a finding consistent with previous UV cross-linking studies identifying an 80-kDa protein as an ARE-interacting protein (Bral and Ramos, 1997). A 42-kDa protein was also predominant in this fraction.…”

Activation profiles of HSPA5 during the glomerular mesangial cell stress response to chemical injury

“…Concentrations of 1 and 10 M HgCl 2 and Pb 2ϩ acetate are within the range that induces ER stress in rat C6 glioma cells (Qian et al 1995(Qian et al , 1999(Qian et al , 2001(Qian et al , 2005a, and consistent with environmental concentrations of these metals (Florea and Busselberg, 2006). Likewise, BaP exposures at 3 M span the range of estimated human exposures 0.171-1.64 g/day (Busbee et al 1990;USEPA 1990;Bral and Ramos 1997). None of the treatment regimens tested, except for 30 M BaP, affect cell viability (data not shown).…”

“…Severe functional deficits, including enzymuria, proteinuria, impaired transport, and depressed glomerular filtration rate, occur at exposures Ͼ50 g/dL (Lilis et al 1968;Baker et al 1980;Hong et al 1980;ATSDR, 2005). As described for the heavy metals, BaP exposures at 3 M span the range of estimated human exposures 0.171-1.64 g/d (Busbee et al 1990;USEPA 1990;Bral and Ramos 1997).…”

Altered Gene Expression Patterns in MCF-7 Cells Induced by the Urban Dust Particulate Complex Mixture Standard Reference Material 1649a

“…PAHs can induce tumor promotion through several, likely combinatorial, mechanisms including negative effects on transcription of tumor-suppressor genes (42), transcriptional activation of proto-oncogenes (43), and activation of enzymes that oxidize procarcinogenic PAHs to reactive metabolites (44). To date, no mutations in the BRCA-1 gene have been identified in sporadic breast cancers, whereas the expression levels of BRCA-1 in breast tumors are lower than those observed in normal mammary tissue (45).…”

Identification of Albumin Precursor Protein, Phi AP3, and α-Smooth Muscle Actin as Novel Components of Redox Sensing Machinery in Vascular Smooth Muscle Cells

“…2A). Two prominent bands of 80 and 100 kDa were present in fraction 6, a finding consistent with previous UV cross-linking studies identifying an 80-kDa protein as an ARE-interacting protein (Bral and Ramos, 1997). A 42-kDa protein was also predominant in this fraction.…”

Activation profiles of HSPA5 during the glomerular mesangial cell stress response to chemical injury

“…Concentrations of 1 and 10 M HgCl 2 and Pb 2ϩ acetate are within the range that induces ER stress in rat C6 glioma cells (Qian et al 1995(Qian et al , 1999(Qian et al , 2001(Qian et al , 2005a, and consistent with environmental concentrations of these metals (Florea and Busselberg, 2006). Likewise, BaP exposures at 3 M span the range of estimated human exposures 0.171-1.64 g/day (Busbee et al 1990;USEPA 1990;Bral and Ramos 1997). None of the treatment regimens tested, except for 30 M BaP, affect cell viability (data not shown).…”

“…Severe functional deficits, including enzymuria, proteinuria, impaired transport, and depressed glomerular filtration rate, occur at exposures Ͼ50 g/dL (Lilis et al 1968;Baker et al 1980;Hong et al 1980;ATSDR, 2005). As described for the heavy metals, BaP exposures at 3 M span the range of estimated human exposures 0.171-1.64 g/d (Busbee et al 1990;USEPA 1990;Bral and Ramos 1997).…”

Altered Gene Expression Patterns in MCF-7 Cells Induced by the Urban Dust Particulate Complex Mixture Standard Reference Material 1649a

“…PAHs can induce tumor promotion through several, likely combinatorial, mechanisms including negative effects on transcription of tumor-suppressor genes (42), transcriptional activation of proto-oncogenes (43), and activation of enzymes that oxidize procarcinogenic PAHs to reactive metabolites (44). To date, no mutations in the BRCA-1 gene have been identified in sporadic breast cancers, whereas the expression levels of BRCA-1 in breast tumors are lower than those observed in normal mammary tissue (45).…”