Transgenic overexpression of regucalcin leads to suppression of thapsigargin- and actinomycin D-induced apoptosis in the testis by modulation of apoptotic pathways

Abstract

SUMMARYRecent evidence suggested the involvement of calcium-binding protein regucalcin (RGN) in testicular apoptosis. Herein, we investigated the role of RGN controlling apoptotic pathways in the testis by using a transgenic rat model overexpressing RGN (Tg-RGN). Seminiferous tubules (SeT) from Tg-RGN and their wild-type (Wt) counterparts were cultured ex vivo in presence or absence of apoptosis inducers thapsigargin (Thap, 10 À7 and 10 À6 M) and actinomycin D (Act D, 0.5 and 1 lg/mL). Expression levels of key regulators of apoptosis in SeT of Tg-RGN and Wt animals were determined by quantitative real-time PCR and Western blot analysis. Measurement of caspase-3 enzymatic activity was included as an end point of apoptosis. Tg-RGN SeT treated with 10 À6 M of Thap or 1 lg/mL of Act D showed a diminished enzymatic activity and gene transcription of caspase-3, along with increased mRNA and protein expression of antiapoptotic Bcl-2. Bcl-2/Bax (antiapoptotic/proapoptotic) protein ratio was also enhanced in these SeT. Although caspase-9 mRNA was increased in the SeT of Tg-RGN treated with Thap, no differences were observed at protein level, and no differences were also found on protein levels of apoptosis-inducing factor. mRNA expression of proapoptotic p53 and p21 was strongly decreased in Tg-RGN SeT treated with Thap (10 À6 M) or Act D (1 lg/mL). These findings demonstrated that RGN suppresses Thap-and Act D-induced apoptosis in SeT by modulating the expression and activity of key apoptotic and antiapoptotic factors. Moreover, results indicate that RGN overexpression protects germ cell from apoptosis induced by noxious stimuli, which could be a relevant mechanism for fertility preservation in situations of oncological treatments.

The protective effect of regucalcin against radiation-induced damage in testicular cells

“…RGN has been shown to suppress apoptosis induced by noxious stimuli [10,12], and an increased expression of RGN in response to radiation was detected in pancreatic cell lines [45]. Therefore, we decided to analyze RGN expression after radiation treatment.…”

“…However, the antiapoptotic/proapoptotic (Bcl-2/Bax) protein ratio is ≈2.2-fold higher in irradiated Tg-RGN relatively to irradiated Wt, which indicates a lower rate of apoptosis in these animals. Bcl-2 has been implicated in the inhibition of p53 upregulation in response to various stimuli [12,62], which may explain the reduced levels of p53, p21, and caspase-9 after irradiation. In addition, Bcl-2 is a recognized repressor of the release of cytochrome c to the cytosol [36], inhibiting the formation of the apoptosome and, consequently, the activation of caspase-9.…”

“…Moreover, it is known that RGN plays an important role in the regulation of cell cycle and apoptosis [10][11][12]59]. Therefore, the expression of several cell cycle and apoptosis modulators was evaluated in Tg-RGN and Wt animals.…”

“…Therefore, the enzymatic activity of caspase-3 has been widely used to measure apoptosis [12,40,44].…”

“…RGN has been indicated as a protein that suppresses apoptosis [10][11][12], and has cytoprotective and antioxidant effects [13][14][15][16][17], protecting cells from noxious stimuli.…”