Parathyroid hormone-related protein regulates integrin α6 and β4 levels via transcriptional and post-translational pathways

Abstract

Parathyroid hormone-related protein (PTHrP) enhances prostate cancer (CaP) growth and metastasis in vivo. PTHrP also increases cell survival and migration, and upregulates pro-invasive integrin α6β4 expression. We used the human CaP cell lines C4-2 and PC-3 as model systems to study the mechanisms via which PTHrP regulates α6β4 levels. We report that PTHrP regulates α6 and β4 levels via a transcriptional pathway; β4 regulation involves the NF-κB pathway. PTHrP also regulates β4 levels at the post-translational level. PTHrP inhibits caspase-3 and -7 activities. Post-translational regulation of β4 by PTHrP is mediated via attenuation of its proteolytic cleavage by these caspases. Since α6 dimerizes with β4, increased β4 levels result in elevated α6 levels. Suppressing β4 using siRNA attenuates the effect of caspase inhibition on apoptosis and cell migration. These results provide evidence of a link between PTHrP, integrin α6β4 levels as a function of caspase activity, and cell survival and migration. Targeting PTHrP in CaP cancer, thereby reversing the effect on caspase activity and α6β4 levels, may thus prove therapeutically beneficial.

Fig. 1

Levels of PTHrP, and the integrin α6 and β4 subunits in PC-3 and C4-2 cells transfected with a PTHrP-expressing construct or with PTHrP siRNA. (A) and (B) mRNA levels of PTHrP, and of the integrin α6 and β4 subunits were measured by reverse transcription/real-time PCR Values are expressed relative to the corresponding control (C) value, set arbitrarily at 1.0. For PC-3 cells, this C value represents the mean±SEM of the values obtained for cells transfected with empty vector (control for PTHrP⁺), NTC siRNA (control for PTHrP siRNA-transfected cells), and empty vector+NTC siRNA (control for PTHrP⁺+PTHrP siRNA-transfected cells). For C4-2 cells, C is the mean±SEM for cells transfected with empty vector (control for PTHrP⁺). Each bar is the mean±SEM of three independent experiments.*=Significantly different from C value (P< 0.001). #=Significantly different from FTHrP⁺ value. (C) and (D) Western blot analysis for PTHrP, and integrins α6 and β4. GAPDH was used as loading control. In (A)-(D), PTHrP+=PTHrP-overexpressing cells; C=control cells; PTHrP si=cells transfected with PTHrP siRNA.

Fig. 2

Integrin α6 and β4 promoter activity in PTHrP-overexpressing and control PC-3 and C4-2 cells. Luciferase activity is expressed relative to that in empty vector-transfected control cells (C), set arbitrarily at 1.00. PTHrP⁺=PTHrP-overexpressing cells. Firefly luciferase activity was normalized to Renilla luciferase activity. Each bar is the mean±SEM of three independent experiments. Values for two independent clones are shown. *=Significantly different from the control value (P<0.001).

Fig. 3

(A) NF-κB activity as a function of PTHrP expression. Luciferase activity is expressed relative to that in control cells (C), set arbitrarily at 1.00 and defined as in Fig. 1. (B) Putative NF-κB sites in the α6 and β4 integrin promoter and site directed mutations of these sites. In the NF-κB consensus sequence, g=guanine, R=purine, Y=pyrimidine, and N=any nucleotide. Mutated nucleotides are underlined. (C)-(E) Promoter activity of the wild-type and mutated integrin α6 (C) and β4 (D) and (E) promoters in PTHrP-overexpressing and control PC-3 and C4-2 cells. Luciferase activity is expressed relative to that in wild-type empty vector-transfected control cells (C), set arbitrarily at 1.00. In (A) and (C)-(E), PTHrP⁺=PTHrP-overexpressing cells; C=control cells; PTHrP si=cells transfected with PTHrP siRNA Firefly luciferase activity was normalized to Renilla luciferase activity. Each bar is the mean±SEM of three experiments for each of two independent clones. WT=wild-type, M=mutated (α6); M1 =mutation of upstream NF-κB site (β4); M2=mutation of downstream NF-κB site (β4); Ml+M2=mutated over both upstream and downstream NF-κB sites (β4). *=Significantly different from the respective empty vector-transfected value (P< 0.001); #=significantly different from the respective wild-type value (P< 0.001).

Fig. 4

Protein half life (t_1/2) of the integrin α6 and β4 subunits in PTHrP-overexpressing and control PC-3 cells. Cells were treated with cycloheximide for the indicated time periods. Empty vector-transfected cells were used as controls. PTHrP⁺=PTHrP-overexpressing cells. Integrin α6 and β4 levels were analyzed by Western blot analysis at the indicated time points. (A and B) Representative Western blots showing decay of the integrin α6 (A) and β4 (B) subunits. GAPDH was used as loading control. (C) and (D) Densitometric analysis of the decay of the integrin α6 and β4. Each point is the mean±SEM of data from three independent experiments. The 0 time-point (at the time of cycloheximide addition) is set at 100%. (E) and (F) Comparison of the half-life of the integrin α6 and β4 subunits in PTHrP-overexpressing and control cells. The values were obtained from the data presented in (C) and (D). *=Significantly different from the vehicle control value (P<0.001).

Fig. 5

Caspase activity in PC-3 and C4-2 cells transfected with a PTHrP-expressing construct or with PTHrP siRNA. (A) and (B) Caspase activity. Cells were treated with the indicated concentrations of doxorubicin (dox) for 16 h (C4-2) or 72 h (PC-3). Caspase activity was measured as described in Materials and methods. The control value (C) is defined as in Fig. 1. Each bar is the mean±SEM of three independent experiments for each of two independent clones (PTHrP⁺) or PTHrP siRNAs *=Significantly different from the respective no doxorubicin (dox) value (P<0.001). #=Significantly different from the respective C value (P<0.001). (C) and (D) Western blot analysis for active caspase-3 levels (17–19 kDa) in cells treated with doxorubicin (0.3 µg/ml). Each figure is representative of three independent experiments. In (A)–(D), PTHrP⁺=PTHrP-overexpressing cells; C=control cells; PTHrP si=cells transfected with PTHrP siRNA.

Fig. 6

Effect of caspase inhibition on the integrin β4 and α6 subunit profiles in PTHrP-overexpressing and control PC-3 cells. Cells were treated with MG132 (10 µM) in the presence (+) or absence (−) of zVAD-fmk. Z-FA-fmk was used as negative control (−). The cells were harvested after 16 h and lysates were prepared for Western blotting. Equal loading was confirmed by re-probing with GAPDH. (B) Ubiquitinated (Ub) integrin β4 levels in PTHrP-overexpressing and control PC-3 cells. PTHrP-overexpressing and control PC-3 cells were co-transfected with an integrin p4-expressing construct and a HA-Ub construct. After transfection, cells were treated with MG132 in the presence (+) or absence (−) of zVAD-fmk. Z-FA-fmk was used as negative control (−). Cells were harvested after 16 h and lysates immunoprecipitated with the anti-integrin β4 antibody. The ubiquitination of integrin β4 was detected using the anti-HA antibody. The blot was stripped and reprobed with the anti-integrin β4 antibody. In (A) and (B), each figure is representative of three independent experiments.

Fig. 7

(A) and (B) Apoptosis and (C) and (D) migration of PC-3 and C4-2 cells transfected with a PTHrP-expressing construct or with PTHrP siRNA (A) and (B) Cells were treated with the indicated concentrations of doxorubicin (dox) for 16 h (C4-2) or 72 h (PC-3). Cell apoptosis and migration were measured as described in Materials and methods. The control value (C) is defined as in Fig. 1. Each bar is the mean±SEM of three independent experiments for each of two independent clones (PTHrP⁺) or PTHrP siRNAs. (A) and (B) * = Significantly different from the respective no doxorubicin (dox) value (P<0.001). #=Significantly different from the respective C value (P<.001). (C) and (D) *=Significantly different from the control value (P<0.001).

Fig. 8

Apoptosis and migration of PTHrP-overexpressing and control PC-3 cells with suppressed integrin β4 expression. Cells were transfected with an siRNA targeting the integrin β4 subunit. NTC siRNA was used as control. (A) Western blot analysis for integrin β4 levels in cells transfected with the integrin β4-targeting or NTC siRNA (B) Apoptosis and (C) cell migration were measured in the presence (+) or absence (−) of zVAD-fmk. Z-FA-fmk was used as negative control (−). Apoptosis was induced by treatment with doxorubicin (0.3 µg/ml). Each bar is the mean±SEM of three independent experiments for each of two independent integrin β4-targeting siRNAs. *=Significantly different from the respective NTC value (P< 0.001); #=significantly different from the -zVAD-fmk value (P< 0.001).