Molecular and Cellular Pharmacology
Mitogen-activated protein kinase 3/mitogen-activated protein kinase 1 activates apoptosis during testicular ischemia–reperfusion injury in a nuclear factor-κB-independent manner

https://doi.org/10.1016/j.ejphar.2008.12.028Get rights and content

Abstract

Nuclear factor kappa-B (NF-κB), mitogen-activated protein kinase3/MAPK1 and MAPK8 are involved in testicular ischemia reperfusion injury (testicular-I/R). NF-κB knock-out mice (KO) subjected to testicular-I/R have a reduced testicular damage, blunted MAPK8 activation and enhanced MAPK3/MAPK1 activity. To better understand the role of MAPK3/MAPK1 up-regulation during testicular-I/R, we investigated the effects of PD98059, an inhibitor of MAPK3/MAPK1, in KO mice during testicular-I/R.

KO and wild-type (WT) animals underwent 1 h testicular ischemia followed by 24 h reperfusion or a sham testicular-I/R. Animals received either PD98059 (5 mg/kg/ip) or its vehicle. MAPK3/MAPK1, BAX, caspase-3 and -9 and TNF-α expression were assessed along with histological examination and an immunostaining for protein of apoptosis.

Testicular-I/R caused a greater increase in MAPK3/MAPK1 in KO than in WT animals in both testes. KO mice had a lower expression of the apoptotic proteins and TNF-α as well as reduced histological damage compared to WT. Immunostaining confirmed the lower expression of BAX in the Leydig cells of KO mice. Administration of PD98059, abrogated MAPK3/MAPK1 expression and slightly reduced TNF-α but did not improve or reverse the histological damage in KO. PD98059 significantly reduced the histological damage in WT mice and markedly reduced the apoptotic proteins in KO and WT mice.

These results suggest that testicular-I/R triggers also a pathway of organ damage involving MAPK3/MAPK1, TNF-α, BAX, caspase-3 and -9 that activates an apoptotic machinery in an NF-κB independent manner. These findings should contribute to better understand testicular torsion-induced damage.

Introduction

Testicular torsion is a pathological condition which affects one out of 4000 males younger than 25 years. In humans, testicular torsion is a medical emergency requiring surgical intervention to counter-rotate the testis and spermatic cord to restore blood flow (Ringdahl and Teague, 2006). Restoration of proper blood supplies after repair testicular torsion, however, can also cause testicular ischemia–reperfusion (testicular-I/R) injury which may impair testicular function and, as a consequence, impact fertility (Ringdahl and Teague, 2006).

The mechanism underlying testicular damage after torsion has yet to be fully elucidate. In a mouse model of testicular-I/R we have previously shown that the mitogen-activated protein kinase (MAPK) family has a role in the pathogenesis of testicular ischemia–reperfusion injury (Minutoli et al., 2005, Antonuccio et al., 2006). MAPKs have been shown to induce the phosphorylation of several transcription factors activating up-stream regulatory elements of inducible genes involved in inflammation and apoptosis (Gaestel et al., 2007, Pimienta and Pascual, 2007). These findings suggest that a block of MAPKs activation might represent a potential therapeutic approach in the treatment of testicular torsion.

Evolutionary conserved nuclear factor-kappaB (NF-κB) is a transcription factor which can be induced in response to environmental changes (Neumann and Naumann, 2007). NF-κB regulates a plethora of cellular pathways and processes including the immune response, inflammation, proliferation, apoptosis and calcium homeostasis (Egan and Toruner, 2006). As such, it represents a potential target for novel therapeutic strategies for a wide variety of diseases.

Indeed, NF-κB has also been proposed to play a role in testicular ischemia (Lysiak et al., 2005) where it has been shown to increase expression of genes whose products mediate inflammatory and immune responses, including Tumour Necrosis Factor (TNF-α). We have shown previously that NF-κB and MAPK pathways interact to produce damage after testicular-I/R (Minutoli et al., 2007).

NF-κB knock-out mice (KO) subjected to testicular-I/R have a reduced testicular damage, blunted MAPK8 (also named JNK) and MAPK14 (also named p-38) activation and enhanced MAPK3/MAPK1 (also named ERK1/2) activity when compared to wild type animals. Since MAPKs are involved the activation of NF-κB (Barnes, 1997), it can be hypothesized that both MAPK8 and MAPK14 are severely blunted in mice lacking the NF-κB gene and may not be involve in KO mice following testicular ischemia–reperfusion. By contrast KO animals subjected to testicular-I/R showed a greater MAPK3/MAPK1 expression than in wild type mice suggesting several hypothetical explanation(s) for this finding: i) MAPK3/MAPK1 might stimulate additional molecular inflammatory pathways other than NF-κB; ii) higher expression of MAPK3/MAPK1 in KO mice might unmask a protective role for this kinase, as already shown for cardioprotection in the so-called reperfusion injury kinase (RISK) pathways (Hausenloy and Yellon, 2004); or iii) augmented MAPK3/MAPK1 expression may serve as an up-stream signal to turn-on a programmed cell death in the testis, as already shown in heart (Dhingra et al., 2007). Activation of the apoptotic machinery has been shown in testicular torsion (Sun et al., 2008), with MAPK3/MAPK1, specifically, triggering apoptosis in several testicular cell lines (Sun et al., 2008).

In the light of these findings, the aim of the present paper was to study the effects of PD98059, an inhibitor of MAPK3/MAPK1, on the expression of apoptotic proteins BAX, caspase 3 and 9 and TNF-α in KO mice during testicular-I/R with the goal of generating a more appropriate approach to the treatment of acute testicular torsion. Positive findings regarding the regulatory pathways and the effect of PD98059 on MAPK3/MAPK1 activation are presented in this experimental model.

Section snippets

Animals and treatment

All procedures complied with the standards for care and use of animal subjects as stated in the Guide for the care and use of Laboratory animals (Institute of Laboratory Animal Resources, National Academy of Sciences, Bethesda, Maryland). Breeding pairs of NF-kappaB KO mice (p105; b6; 129-Nfkb1fm1Bal; KO) and normal control littermates WT mice (C57Bl/6; 129; WT), both groups weighing between 22 and 30 g, were purchased from the Jackson Laboratory (Bar Harbor, Maine). The animals were provided a

MAPK3/MAPK1 expression in testicular-I/R: effects of PD98059, an inhibitor of MAPK3/MAPK1, in KO and WT mice

Testicular ischemia–reperfusion injury produced a greater increase of phosphorylated MAPK3/MAPK1 (p-MAPK3/MAPK1) in KO mice than in WT animals (Fig. 1) after 30 min of reperfusion. This higher expression was evidenced in the ipsilateral testis and contralateral one (Fig. 1). No changes were observed in the total form of this kinase in both testes of either KO and WT mice (Fig. 1). Administration of PD98059, an inhibitor of MAPK3/MAPK1, immediately after the beginning of reperfusion, blunted the

Discussion

Torsion of the spermatic cord seriously threatens the continued viability of the ipsilateral testis. In addition function of the contralateral testis may be also damaged by unilateral torsion. The two most important factors determining testicular damage are the degree of twisting and the time period between injury and surgical repair to counter-rotate both testis and spermatic cord for inducing reperfusion. Besides resolving the acute ischemia, the surgical procedure of testicular detorsion

Acknowledgements

The authors are grateful to Dr. Bruce Burnett for language revision.

References (28)

  • AkcoraB. et al.

    The protective effect of darbepoetin alfa on experimental testicular torsion and detorsion injury

    Int. J. Urol

    (2007)
  • AksoyH. et al.

    Dehydroepiandrosterone treatment attenuates reperfusion injury after testicular torsion and detorsion in rats

    J. Pediatr. Surg.

    (2007)
  • AntonuccioP. et al.

    Lipid peroxidation activates mitogen-activated protein kinases in testicular ischemia-reperfusion injury

    J. Urol.

    (2006)
  • BarnesP.J.

    Nuclear factor-kappa B

    Int. J. Biochem. Cell Biol.

    (1997)
  • BeheshtianA. et al.

    Protective effects of sildenafil administration on testicular torsion/detorsion damage in rats

    World J. Urol.

    (2008)
  • DhingraS. et al.

    p38 and ERK1/2 MAPKs mediate the interplay of TNF-alpha and IL-10 in regulating oxidative stress and cardiac myocyte apoptosis

    Am. J. Physiol. Heart Circ. Physiol.

    (2007)
  • DokmeciD.

    Testicular torsion, oxidative stress and the role of antioxidant therapy

    Folia Med. (Plovdiv)

    (2006)
  • EganL.J. et al.

    NF-kappaB signaling: pros and cons of altering NF-kappaB as a therapeutic approach

    Ann. N. Y. Acad. Sci.

    (2006)
  • GaestelM. et al.

    Protein kinases as small molecule inhibitor targets in inflammation

    Curr. Med. Chem.

    (2007)
  • HausenloyD.J. et al.

    New directions for protecting the heart against ischemia–reperfusion injury: targeting the reperfusion injury salvage kinase (RISK) pathway

    Cardiovasc. Res.

    (2004)
  • KyriakisJ.M. et al.

    Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation

    Physiol. Rev.

    (2001)
  • LysiakJ.J.

    The role of tumor necrosis factor-alpha and interleukin-1 in the mammalian testis and their involvement in testicular torsion and autoimmune orchitis

    Reprod. Biol. Endocrinol.

    (2004)
  • LysiakJ.J. et al.

    Essential role of neutrophils in germ cell-specific apoptosis following ischemia/reperfusion injury of the mouse testis

    Biol. Reprod.

    (2001)
  • LysiakJ.J. et al.

    Ischemia–reperfusion of the murine testis stimulates the expression of proinflammatory cytokines and activation of c-jun N-terminal kinase in a pathway to E-selectin expression

    Biol. Reprod.

    (2003)
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