UCHL1 Regulates Radiation Lung Injury via Sphingosine Kinase-1

Abstract

GADD45a is a gene we previously reported as a mediator of responses to acute lung injury. GADD45a-/- mice express decreased Akt and increased Akt ubiquitination due to the reduced expression of UCHL1 (ubiquitin c-terminal hydrolase L1), a deubiquitinating enzyme, while GADD45a-/- mice have increased their susceptibility to radiation-induced lung injury (RILI). Separately, we have reported a role for sphingolipids in RILI, evidenced by the increased RILI susceptibility of SphK1-/- (sphingosine kinase 1) mice. A mechanistic link between UCHL1 and sphingolipid signaling in RILI is suggested by the known polyubiquitination of SphK1. Thus, we hypothesized that the regulation of SphK1 ubiquitination by UCHL1 mediates RILI. Initially, human lung endothelial cells (EC) subjected to radiation demonstrated a significant upregulation of UCHL1 and SphK1. The ubiquitination of EC SphK1 after radiation was confirmed via the immunoprecipitation of SphK1 and Western blotting for ubiquitin. Further, EC transfected with siRNA specifically for UCHL1 or pretreated with LDN-5744, as a UCHL1 inhibitor, prior to radiation were noted to have decreased ubiquitinated SphK1 in both conditions. Further, the inhibition of UCHL1 attenuated sphingolipid-mediated EC barrier enhancement was measured by transendothelial electrical resistance. Finally, LDN pretreatment significantly augmented murine RILI severity. Our data support the fact that the regulation of SphK1 expression after radiation is mediated by UCHL1. The modulation of UCHL1 affecting sphingolipid signaling may represent a novel RILI therapeutic strategy.

Keywords: UCHL1; radiation lung injury; sphingosine kinase 1.

Figure 1

Radiation induces lung EC UCHL1 and SphK1 expression in vitro. Human pulmonary artery ECs were subjected to single-dose irradiation (10 or 20 Gy) for 4 or 24 h, and lysates were then subjected to Western blotting for SphK1 and UCHL1. (A) Representative blots shown with the same loading controls used for both SphK1 and UCHL1. (B) Densitometry from multiple experiments was performed for SphK1 and UCHL1, and both normalized to actin (n > 3 per condition, * p < 0.05 compared to control cells).

Figure 2

Radiation-induced SphK1 ubiquitination is regulated by UCHL1. (A) Human pulmonary artery ECs were subjected to radiation (IR, 20 Gy, 6 h) prior to immunoprecipitation with a SphK1 antibody followed by Western blotting for ubiquitin with either an anti-mouse secondary antibody (left panel) or HRP-conjugated protein A (middle panel). Whole-cell lysates from control cells were used for Western blotting for SphK1 (right panel) to confirm a predominant SphK1 band density at 50 kD. In separate experiments, human pulmonary artery ECs were (B) transfected with siRNA specific for UCHL1 (siUCHL1, 100 nM, 3 d) or non-specific siRNA (nsRNA) or (C) treated with LDN (5 µM, 1.5 h) prior to irradiation (20 Gy, 6 h) followed by the immunoprecipitation of SphK1 and Western blotting for ubiquitin. Representative blots shown. Densitometry quantified and expressed as ubq-SphK1 was normalized to total SphK1 (n = 3 per condition, * p < 0.05).

Figure 3

Expression levels of UCHL1 and SphK1 are interdependent in lung ECs. (A) Human pulmonary artery ECs were transfected with UCHL1 siRNA (100 nM, 3 d) or a UCHL1 overexpression vector (3 d) prior to Western blotting for SphK1. (B) In separate experiments, human pulmonary artery ECs were treated with a pharmacologic SphK1 inhibitor, PF-543 (10 µM), 1 h prior to irradiation, followed by Western blotting for UCHL1. Representative blots are shown.

Figure 4

Lung EC barrier enhancement by tyspinate, an S1P analog, is attenuated by radiation and UCHL1 inhibition. (A) Human pulmonary artery ECs were transfected with siRNA specific for UCHL1 (siUCHL1, 100 nm, 3 d) or non-specific RNA (nsRNA) and grown to confluence on gold-plated microelectrodes prior to irradiation (20 Gy, 4 h) followed by treatment with tyspinate (Tys, 1 μM) and the measurement of transendothelial electrical resistance (TER). (B) In similar experiments, cells were subjected to irradiation (20 Gy, 4 h) prior to treatment with LDN (5 µM, 1.5 h) or a vehicle followed by tysipinate (Tys, 1 μM) (* p < 0.05, n = 3/experimental condition).

Figure 5

Radiation induces lung UCHL1 expression in vivo. (A) Male wild-type (WT) mice were subjected to RILI (20 Gy), and lungs were then harvested and used for RT-PCR to assess mRNA levels of UCHL1 at 6 wks (n = 3/group, * p < 0.05 compared to controls). (B) Lungs from RILI-challenged (20 Gy) WT mice at 6 wks were subjected to Western blotting for UCHL1 (n = 3 animals/group, * p < 0.05 compared to controls).

Figure 6

UCHL1 inhibition exacerbates RILI. WT mice were treated with LDN (5 mg/kg, IP) or a vehicle at the time of irradiation (IR, 20 Gy) and then 3x/wk post-radiation for 6 wks. BAL fluid was then collected and assessed for total protein (A) and total cell counts (B) (n ≥ 3/group, * p < 0.05 compared to respective controls, * p < 0.05).

Figure 7

Proposed role of UCHL1 in sphingolipid-mediated responses to radiation. Our data are consistent with UCHL1-mediated EC barrier protection in response to radiation via decreased SphK1 ubiquitination which, in turn, promotes the increased activation of sphingosine 1-phosphate (S1P) as well as further increases in UCHL1 expression levels, potentially through effects on histone acetylation and DNA methylation. S1P is known to signal through S1P receptors on the surface of EC (S1PR1-3) to promote the activation of the small GTPase Rac1, actin cytoskeletal rearrangement, and EC barrier enhancement.