Regulation of amyloid-β levels by matrix metalloproteinase-2/9 (MMP2/9) in the media of lung cancer cells

Abstract

In this study, we set out to identify regulators of intact amyloid-β40/42 (Aβ) levels in A549 (p53 wild-type) and H1299 (p53-null) lung cancer cell media. Higher Aβ levels were detected in the media of A549 than H1299 cells without or with treatment with 4-methylumbelliferone (4-MU) and/or the anti-CD44 antibody (5F12). Using inhibitors, we found that PI3K, AKT, and NFκB are likely involved in regulating Aβ levels in the media. However, increased Aβ levels that more closely resembled those found upon 4-MU co-treatment resulted from MMP2/9 inhibition, suggesting that MMP2/9 maybe the main contributors to regulation of Aβ levels in the media. Differences in Aβ levels might be accounted for, in part, by p53 since blocking p53 function in A549 cells resulted in decreased Aβ levels, increased MMP2/9 levels, increased PI3K/AKT activities and the phospho/total NFκB ratio. Using siRNA targeted against MMP2 or MMP9, we found increased Aβ levels in the media, however, MMP2 knockdown led to Aβ levels closely mimicking those detected by co-treatment with 4-MU. Cell viability or apoptosis upon treatment with either MMP2 or MMP9 siRNA along with Aβ immunodepletion, showed that MMP2 is the predominant regulator of the cytotoxic effects induced by Aβ in lung cancer cells.

Differences in the Relative Abundance of ProBDNF and Mature BDNF in A549 and H1299 Human Lung Cancer Cell Media

“…We then found that ATP, thought to reduce misfolding of amyloid beta, strengthened interactions between amyloid beta and humanin [57]. More recently, we showed that higher intact amyloid beta 40/42 levels are present in the media of A549 (p53 wild-type) than in H1299 (p53-null) lung cancer cell media [44].…”

“…Opposing functional roles have been attributed to secreted BDNF and proBDNF on cell proliferation and apoptosis [6,8]. In view of the differences we found in the intact levels of amyloid beta 40/42 in the conditioned media of lung cancer cell lines, [44] we asked whether there are also differences in the levels of proBDNF and mBDNF in the media of these cells. Our results show that proBDNF levels are higher in A549 (p53-positive) cell media than in the media of H1299 (p53-null) cells (Figure 2).…”

“…In accordance with previous findings showing that NFκB and p53 have opposing effects in cancer cells with antagonistic signaling, crossregulating each other's activity and suppressing each other's ability to enhance gene expression, [18] our observation that NFκB phosphorylation is increased by inhibiting p53 (Figure 5C) suggests that p53 acts as an antagonist of NFκB phosphorylation in A549 cells. In examining the levels of amyloid beta, we have previously found that treatment with the p53 inhibitor, pifithrin-α, led to higher levels of MMP2 and MMP9 in A549 cellconditioned media, while no effects were observed on either MMP2 or MMP9 levels with this treatment in the p53-null H1299 cell media, suggesting that p53 functions to decrease MMP2/9 levels in the media of A549 cells [44]. These results are consistent with our findings in this study (Figure 4).…”

“…Cells were grown in 10% FBS-supplemented media for 24 h. The following day, the cell monolayers were incubated in serum-free media for 24 h and then treated as indicated for 72 h with the inhibitors, as described in the Methods Section and as we recently reported [44]. The media were collected, and then the same amount of protein of each sample was used to quantitate mBDNF and proBDNF (Methods).…”

Interactions of Amyloid-β with Membrane Proteins

“…In a non-pathological process, mainly shorter Aβ 40 is produced, which is usually about 80–90%. The longer form of Aβ 1–42 is more hydrophobic and fibrillogenic [ 22 , 23 ].…”