Influence of Anoctamin-4 and -9 on ADAM10 and ADAM17 Sheddase Function

Abstract

Ca2+-activated Cl− channels (TMEM16, also known as anoctamins) perform important functions in cell physiology, including modulation of cell proliferation and cancer growth. Many members, including TMEM16F/ANO6, additionally act as Ca2+-activated phospholipid scramblases. We recently presented evidence that ANO6-dependent surface exposure of phosphatidylserine (PS) is pivotal for the disintegrin-like metalloproteases ADAM10 and ADAM17 to exert their sheddase function. Here, we compared the influence of seven ANO family members (ANO1, 4, 5, 6, 7, 9, and 10) on ADAM sheddase activity. Similar to ANO6, overexpression of ANO4 and ANO9 led to increased release of ADAM10 and ADAM17 substrates, such as betacellulin, TGFα, and amphiregulin (AREG), upon ionophore stimulation in HEK cells. Inhibitor experiments indicated that ANO4/ANO9-mediated enhancement of TGFα-cleavage broadened the spectrum of participating metalloproteinases. Annexin V-staining demonstrated increased externalisation of PS in ANO4/ANO9-overexpressing cells. Competition experiments with the soluble PS-headgroup phosphorylserine indicated that the ANO4/ANO9 effects were due to increased PS exposure. Overexpression of ANO4 or ANO9 in human cervical cancer cells (HeLa), enhanced constitutive shedding of the growth factor AREG and increased cell proliferation. We conclude that ANO4 and ANO9, by virtue of their scramblase activity, may play a role as important regulators of ADAM-dependent cellular functions.

Scramblases as Regulators of Proteolytic ADAM Function

“…In cells overexpressing ANO6, however, the substrate release could not be blocked anymore with ADAM17 inhibitors but only with broad-spectrum metalloprotease inhibitors, indicating that further metalloproteases participated in the cleavage of TGF-alpha [30]. Recently, we obtained similar results upon ANO4 and ANO9 overexpression [34]. Could other membrane-anchored proteases or proteins operating at the cell surface underlie similar regulatory principles?…”

“…We summarize current knowledge regarding the significance of PS externalization on proteolytic activity of ADAM10 and ADAM17. Arguments are presented to support the concept that scramblase-mediated shuffling of phospholipids is a key step leading to ADAM10 and ADAM17 activation [30][31][32][33][34][35]. The potential functional consequences of these interactions are discussed and future challenges to be met in field are outlined.…”

“…In an immediate context, 260 both ADAMs play a profound role in many types of cancers [89,90]. Recent results from 261 our working group indicate a direct link between ANO4 and ANO9 scramblase activity 262 and ADAM function [34]. Overexpression of ANO4 and ANO9 led to increased release of 263 ADAM10 and ADAM17 substrates, such as betacellulin, TGF-α, and AREG, upon iono-264 phore stimulation in HEK cells.…”

ANO4 Expression Is a Potential Prognostic Biomarker in Non-Metastasized Clear Cell Renal Cell Carcinoma

“…Leitzke et al reported ANO4 to modulate disintegrin-like metalloproteases ADAM 10 and 17 sheddase activity, evident through the increasing ADAM10 and 17 substrates: transforming growth factor alpha (TGF- α ), amphiregulin (AREG), and betacellulin. They also demonstrated that the effects brought upon by the overexpression of ANO4 are due to it is scramblase activity, consequently resulting in diminishing AREG and increased cellular proliferation [ 75 ]. Maniero et al recognized ANO4 as a significant gene expressed in the zona glomerulosa cells despite its contradictory effects on aldosterone secretion [ 76 , 77 ].…”

Overcoming the tumor biological barrier: a multistage drug delivery strategy for DDR1-mediated resistant colorectal cancer therapy

“…The impact of ANO family members on A Disintegrin and Metalloproteinases (ADAMs) sheddase activity has been proved 22,23 . ADAM10 and 17 could shed numerous membrane-bound proteins, releasing their ectodomains 24 .…”