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

Area of Science:

• Cell Biology
• Molecular Biology
• Cancer Research

Background:

• Calcium-activated chloride channels (TMEM16/anoctamins) are crucial for cell physiology, including proliferation and cancer.
• Some TMEM16 family members, like TMEM16F/ANO6, function as calcium-activated phospholipid scramblases.
• ANO6-dependent phosphatidylserine (PS) exposure is vital for ADAM10 and ADAM17 sheddase activity.

Purpose of the Study:

• To investigate the influence of seven ANO family members (ANO1, 4, 5, 6, 7, 9, 10) on ADAM sheddase activity.
• To determine if ANO4 and ANO9, similar to ANO6, modulate ADAM-dependent sheddase functions.
• To explore the role of ANO4 and ANO9 in cancer cell proliferation and growth factor shedding.

Main Methods:

• Overexpression of ANO family members in HEK cells.
• Analysis of ADAM10 and ADAM17 substrate release (betacellulin, TGFα, amphiregulin).
• Annexin V staining to assess phosphatidylserine (PS) externalization.
• Inhibitor experiments and competition assays with phosphorylserine.
• Assessment of AREG shedding and cell proliferation in HeLa cancer cells.

Main Results:

• Overexpression of ANO4 and ANO9 increased the release of ADAM10 and ADAM17 substrates.
• ANO4 and ANO9 enhanced TGFα-cleavage, broadening the spectrum of involved metalloproteinases.
• ANO4 and ANO9 overexpression led to increased PS externalization.
• ANO4 and ANO9 enhanced constitutive shedding of amphiregulin (AREG) and increased cell proliferation in HeLa cells.

Conclusions:

• ANO4 and ANO9, through their scramblase activity, increase phosphatidylserine (PS) exposure.
• This PS exposure enhances the activity of ADAM metalloproteinases.
• ANO4 and ANO9 may act as significant regulators of ADAM-dependent cellular functions, including cancer cell proliferation.