Signal transduction in CHO cells stably transfected with domain-selective forms of murine ACE

Abstract

Membrane-bound human angiotensin-converting enzyme (ACE) has been reported to initiate intracellular signaling after interaction with substrates or inhibitors. Somatic ACE is known to contain two distinct, extracellular catalytic centers. We analyzed the signal transduction mechanisms in cells transfected with different forms of murine ACE (mACE) and investigated whether the two domains are similarly involved in these processes. For this purpose, CHO cells were stably transfected with mACE or with its domain-selective mutants. In addition to these modified cellular models, human umbilical vein endothelial cells were used in this study. Signal transduction molecules such as JNK and c-Jun were analyzed after activation of cells with several ACE substrates and inhibitors. ACE-targeting compounds such as substrates, inhibitors, or even the ACE product angiotensin-II induce in mACE-expressing cells a signal transduction response. These processes are also evoked by partially inactivated forms of mACE and finally result in an enhanced cyclooxygenase-2 transcription. Surprisingly, the membrane-bound ACE activity is also influenced by ACE-targeted interventions. Our data suggest that the two catalytic domains of mACE do not function independently but that the signal transduction is influenced by negative cooperativity of the two catalytic domains. This study underlines that ACE indeed has receptor-like properties which occur in a species-specific manner.

Angiotensin II Binding to Angiotensin I–Converting Enzyme Triggers Calcium Signaling

“…Moreover, we could demonstrate that these effects are additive, different from the results shown by Sun et al, 18 who 22,23 Ang II may also be equally effective at both sites. However, the capacity of binding to the enzyme is not the sole property of a molecule influencing its ability to activate ACE.…”

“…The activation of the c-Jun N-terminal kinase in CHO cells was shown to be promoted not only exclusively by the active C-domain of human ACE 17 but also by the N-domain in murine ACE. 18 We could show that the calcium signaling promoted by Ang II in these cells is produced to a similar extent by both domains of human ACE, and the interaction of these compounds with a single catalytic domain is obviously sufficient to induce the signaling effect.…”

“…5,16 Interestingly, neither Ang I nor its product, Ang II, was shown to be able to elicit this signaling pathway. 17 Recently, Sun et al 18 showed that Ang II could induce a c-Jun N-terminal kinase activation via murine ACEtransfected cells and also bind to ACE. In this study, we describe for the first time that Ang II is also capable of triggering a calcium signaling pathway, different from that described by Fleming and others.…”

The binding of captopril to angiotensin I-converting enzyme triggers activation of signaling pathways

“…Literature data showed that some ACE substrates or inhibitors triggered an increase of casein kinase 2 (CK2) activity, leading to ACE Ser1270 residue phosphorylation with subsequent activation of JNK, as well as the accumulation of phosphorylated c-Jun in the nuclei of endothelial cells (37). Similar results were also obtained for murine ACE (61). It also has been demonstrated that ANG II is able to interact with ACE in Chinese hamster ovary (CHO) cells transfected with this enzyme and melanoma cells, triggering calcium signaling and promoting an increase of reactive oxygen species by the activation of the enzyme (24).…”

“…As described above, several studies have shown that binding of ACE inhibitors to the enzyme triggers signaling pathway activation (3,24,37,61). However, to date, none of these studies were conducted with a sulfhydryl group containinginhibitor such as captopril, and none of them have investigated ERK1/2 phosphorylation signaling pathway either.…”