Role of hypoxia-induced transglutaminase 2 in pulmonary artery smooth muscle cell proliferation

Abstract

We previously reported that transglutaminase 2 (TG2) activity is markedly elevated in lungs of hypoxia-exposed rodent models of pulmonary hypertension (PH). Since vascular remodeling of pulmonary artery smooth muscle cells (PASMCs) is important in PH, we undertook the present study to determine whether TG2 activity is altered in PASMCs with exposure to hypoxia and whether that alteration participates in their proliferative response to hypoxia. Cultured distal bovine (b) and proximal human (h) PASMCs were exposed to hypoxia (3% O2) or normoxia (21% O2). mRNA and protein expression were determined by PCR and Western blot analyses. TG2 activity and function were visualized and determined by fluorescent labeled 5-pentylamine biotin incorporation and immunoblotting of serotonylated fibronectin. Cell proliferation was assessed by [(3)H]thymidine incorporation assay. At 24 h, both TG2 expression and activity were stimulated by hypoxia in bPASMCs. Activation of TG2 by hypoxia was blocked by inhibition of the extracellular calcium-sensing receptor or the transient receptor potential channel V4. In contrast, TG2 expression was blocked by inhibition of the transcription factor hypoxia-inducible factor-1α, supporting the presence of separate mechanisms for stimulation of activity and expression of TG2. Pulmonary arterial hypertension patient-derived hPASMCs were found to proliferate significantly more rapidly and respond to hypoxia more strongly than control-derived hPASMCs. Similar to bovine cells, hypoxia-induced proliferation of patient-derived cells was blocked by inhibition of TG2 activity. Our results suggest an important role for TG2, mediated by intracellular calcium fluxes and HIF-1α, in hypoxia-induced PASMC proliferation and possibly in vascular remodeling in PH.

Keywords: CaSR; HIF-1α; TG2; TRPV4; pulmonary artery smooth muscle cells.

Fig. 1.

Transglutaminase 2 (TG2) mRNA, protein expression, and activity are increased in bovine (b) pulmonary artery smooth muscle cells (PASMCs) exposed to hypoxia. A: RT-PCR analysis showing TG2 mRNA in serum-starved bPASMCs exposed to 24-h normoxia (21% O₂) or hypoxia (3% O₂) (representative images of PCR products shown in duplicates). B: representative Western blots of cell extracts demonstrating TG2 protein expression under normoxia and hypoxia conditions for 2–24 h. C: bar graph demonstrating the effect of hypoxia on TG2 protein expression measured by densitometry analysis (n = 3 blots/condition). TG2 protein expression (78 kDa) was detected with anti-TG2 antibody. Equal amounts of RNA and protein were loaded as indicated by the loading control GAPDH and smooth muscle α-actin (42 kDa). D: representative images showing intracellular TG2 activity detected by 5-(biotinamido)pentylamine (5-BP) assay and imaged on a microscope with an oil immersion ×40 objective lens. Serum starved bPASMCs cultured on coverslips were incubated with 5-BP (400 μM) under normoxia or hypoxia conditions for 2 and 24 h. The 5-BP incubation step was omitted in control cells. Streptavidin AlexaFluor555-conjugate and DAPI counterstain were used to stain intracellular TG2 activity (red) and nucleus (blue), respectively. E: bar graph showing changes in TG2 activity assessed by measuring the relative staining intensity per cell with ImageJ software. At least 2 slides/condition were used (n = 10 images/condition). *P < 0.05, significantly different from normoxia control. #P < 0.05, significantly different from hypoxia control. ns, Not significant.

Fig. 2.

Hypoxia increases serotonylation of fibronectin that is blocked by TG2 inhibitor ERW1041E. A: serum-starved bPASMCs were exposed to normoxia (21% O₂) or hypoxia (3% O₂) for 24 h. Western blots of cell extracts showing the effect of normoxia and hypoxia on serotonylated fibronectin (sFn) and fibronectin (Fn) levels. B: bar graph demonstrating changes in TG2 activity assessed by measuring sFn normalized to Fn levels by densitometry analysis of blots in A (n = 5 blots/condition). C: serum-starved bPASMCs were pretreated for 1 h with vehicle or ERW1041E and exposed to hypoxia for 24 h. Representative Western blots showing the effect of ERW1041E on TG2 expression and activity (sFn). D: bar graph demonstrating the effect of ERW1041E on sFn/Fn ratio measured by densitometry analysis (n = 3 blots/treatment group). sFN (220 kDa) was detected with anti-5-HT antiserum. Fn (220 kDa) was detected by use of anti-Fn antibody. TG2 protein expression (78 kDa) was detected with anti-TG2 antibody. Smooth muscle α-actin (42 kDa) was blotted on the stripped membrane as loading control. *P < 0.05, significantly different from normoxia control. #P < 0.05, significantly different from vehicle-treated hypoxia control.

Fig. 3.

Hypoxia-induced bPASMC proliferation is blocked by TG2 inhibitor ERW1041E and TG2 cross-linking defective mutant C277V. A: serum-starved bPASMCs were pretreated with TG2 inhibitor ERW1041E for 1 h and exposed to normoxia (21% O₂) or hypoxia (3% O₂) for 24 h. B: bPASMCs were either mock transfected or transfected with pcDNA vector alone or with TG2 point mutants, the GTP-binding defective R580L, and the cross-linking defective C277V. Control cells were treated with transfection reagent alone. The next day bPASMCs were serum starved and then exposed to normoxia or hypoxia for 24 h. Cell proliferation was quantified by [³H]thymidine incorporation assay and expressed as cell counts (n = 12 wells/treatment group). *P < 0.05, significantly different from vehicle-treated or mock-transfected normoxia control. #P < 0.05, significantly different from vehicle-treated or mock-transfected hypoxia control.

Fig. 4.

Influence of CaSR and TRPV4 inhibitors on hypoxia-induced TG2 activity and expression. Serum-starved bPASMCs were pretreated for 1 h with vehicle or CaSR inhibitor (NPS2390) or TRPV4 inhibitor (HC-067047) and exposed to hypoxia (3% O₂) conditions for 24 h. Representative Western blots showing the effect of NPS2390 (A) and HC-067047 (C) on hypoxia-induced TG2 activity (sFn) and expression of Fn and TG2. Bar graph demonstrating the effect of NPS2390 (B) and HC-067047 (D) on sFn/Fn ratio measured by densitometry analysis (n = 4 blots/treatment group). sFN (220 kDa) was detected with anti-5-HT antiserum. Fn (220 kDa) was detected by using anti-Fn antibody. TG2 protein expression (78 kDa) was detected with anti-TG2 antibody. Smooth muscle α-actin (42 kDa) was blotted on the stripped membrane as loading control. #P < 0.05, significantly different from vehicle-treated hypoxia control.

Fig. 5.

TG2 expression is blocked by HIF-1α inhibitor, PX-478. A: representative Western blots demonstrating the effect of cobalt chloride (CoCl₂; 300 μM) and vehicle on HIF-1α and TG2 expression under normoxia (21% O₂) or hypoxia (3% O₂) conditions at 24 h. B: bar graph demonstrating the effect of CoCl₂ and hypoxia on HIF-1α and TG2 expression measured by densitometry analysis (n = 4 blots/treatment group). C: representative Western blots demonstrating the effect of vehicle and PX-478 in presence of CoCl₂ for 24 h. Bar graph demonstrating the effect of PX-478 on CoCl₂-induced expression of HIF-1α (D) and TG2 (E) measured by densitometry analysis (n = 4 blots/treatment group). F: representative Western blots demonstrating the effect of vehicle or PX-478 under hypoxia for 24 h. G: bar graph demonstrating the effect of PX-478 on hypoxia-induced TG2 expression measured by densitometry analysis (n = 4 blots/treatment group). HIF-1α (120 kDa) was detected by immunoblotting with anti-HIF-1α antibody. TG2 protein expression (78 kDa) was detected with anti-TG2 antibody. Smooth muscle α-actin (42 kDa) was blotted on the stripped membrane as loading control. *P < 0.05, significantly different from vehicle-treated normoxia control. #P < 0.05, significantly different from CoCl₂-treated control or vehicle-treated hypoxia control.

Fig. 6.

Effect of PX-478 and ERW1041E on TG2 and HIF-1α activity. A: representative Western blots shows the effect of vehicle and HIF-1α inhibitor PX-478 in presence of CoCl₂ for 24 h. Bar graph demonstrating the effect of PX-478 on sFn (B) and Fn (C) expression measured by densitometry analysis (n = 3 blots/treatment group). D: representative Western blots shows the effect of vehicle or TG2 inhibitor ERW1041E in presence of CoCl₂ for 24 h. E: bar graph demonstrating the effect of ERW1041E on HIF-1α expression measured by densitometry analysis (n = 4 blots/treatment group). HIF-1α (120 kDa) was detected by immunoblotting with anti-HIF-1α antibody. sFN (220 kDa) was detected with anti-5-HT antiserum. Fn (220 kDa) was detected with anti-Fn antibody. Smooth muscle α-actin (42 kDa) was blotted on the stripped membrane as loading control. *P < 0.05, significantly different from vehicle-treated normoxia control. #P < 0.05, significantly different from CoCl₂-treated control.

Fig. 7.

Hypoxia-induced pulmonary arterial hypertension (PAH) human (h)PASMC proliferation is reduced with TG2 inhibitor ERW1041E. A: control subject and PAH patient-derived hPASMCs were exposed to normoxia (21% O₂) or hypoxia (3% O₂) for 24 h. B: PAH hPASMCs were pretreated with ERW1041E or vehicle for 1 h and exposed to normoxia or hypoxia for 24 h. Cell proliferation was quantified by [³H]thymidine incorporation assay and expressed as cell counts (n = 8 wells/treatment group). *P < 0.05, significantly different from vehicle-treated normoxia control. #P < 0.05, significantly different from vehicle-treated hypoxia control.

Fig. 8.

Scheme of hypoxia-induced TG2 expression and activity in PASMCs.