The underlying mechanism of proinflammatory NF-κB activation by the mTORC2/Akt/IKKα pathway during skin aging

Abstract

Mammalian target of rapamycin complex 2 (mTORC2), one of two different enzymatic complexes of mTOR, regulates a diverse set of substrates including Akt. mTOR pathway is one of well-known mediators of aging process, however, its role in skin aging has not been determined. Skin aging can be induced by physical age and ultraviolet (UV) irradiation which are intrinsic and extrinsic factors, respectively. Here, we report increased mTORC2 pathway in intrinsic and photo-induced skin aging, which is implicated in the activation of nuclear factor-κB (NF-κB). UVB-irradiated or aged mice skin revealed that mTORC2 activity and its component, rictor were significantly upregulated which in turn increased Akt activation and Akt-dependent IκB kinase α (IKKα) phosphorylation at Thr23 in vivo. We also confirmed that UVB induced the mTORC2/Akt/IKKα signaling pathway with HaCaT human normal keratinocytes. The increased mTORC2 signaling pathway during skin aging were associated to NF-κB activation. Suppression of mTORC2 activity by the treatment of a mTOR small inhibitor or knockdown of RICTOR partially rescued UVB-induced NF-κB activation through the downregulation of Akt/IKKα activity. Our data demonstrated the upregulation of mTORC2 pathway in intrinsic and photo-induced skin aging and its role in IKKα/NF-κB activation. These data not only expanded the functions of mTOR to skin aging but also revealed the therapeutic potential of inhibiting mTORC2 in ameliorating both intrinsic skin aging and photoaging.

Keywords: Gerotarget; NF-κB; UV; mTORC2; skin aging.

Figure 1. Changes in the expression and activity of mTORC2 in aged skin

Western blotting was performed to detect the levels of mTOR phosphorylated at Ser2481, Rictor (A and B), and activated Akt at Thr308 and Ser473 (C and D) in skin homogenates of 12- and 24-month-old mice (A and C) and in skin homogenates of control and UVB-irradiated mice (B and D). The blots were quantified by densitometry. The blots of phospho-mTOR (Ser2481) and Rictor were normalized to β-actin and the phosphorylated forms of Akt were normalized to total Akt. Bars represent the mean percentage value ± SEM in 12-month-old mice (n = 7-8, * p < 0.05, ** p < 0.01 vs. 12-month-old mice) and control mice (n = 7-8, * p < 0.05, ** p < 0.01 vs. control mice).

Figure 2. The levels of IKKα and p65 phosphorylation during skin aging

Western blotting was performed to detect the levels of IKKα phosphorylated at Thr23 and p65 at Ser536 in skin homogenates from 12- and 24-month-old mice (A) and in skin homogenates of control and UVB-irradiated mice (B) Blots were quantified by densitometry and normalized to β-actin or TFIIB. Bars represent the mean percentage value ± SEM in 12-month-old mice (n = 7-8, * p < 0.05, ** p < 0.01 vs. 12-month-old mice) and control mice (n = 7-8, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control mice).

Figure 3. The activation of mTORC2 and its downstream signaling by UVB irradiation in HaCaT cells

HaCaT cells were incubated for 15 min - 6 h after UVB irradiation (30 mJ/cm²) (A) Western blotting was performed to detect mTORC2, activation of the Akt signaling pathway, as indicated by the presence of phosphorylated Akt and changes in the IKKαβ/IκBα signaling pathway in the cytosolic fraction. Nuclear phosphor-IKKα/β (Thr23), phosphor-p65 (Ser536), and total p65 were measured by Western blotting using specific antibodies. To clarify whether Akt regulates IKKα activity, HaCaT cells were treated with 1 μM of the Akt inhibitor Akti-1/2, 1 h prior to UVB irradiation (60 mJ/cm²) (B). Two hours after UVB irradiation Western blotting was performed to detect changes in the signaling pathways and the activation of p65 in the cytosolic and nuclear fractions. β-Actin, histone H1, and TFIIB blots were shown to clarify the same amount of protein loaded in cytosolic and nuclear fractions. For each protein, one representative blot is shown from 3 experiments that yielded similar results.

Figure 4. Attenuation of UVB-induced p65 activation by mTORC2 inhibition in HaCaT cells

HaCaT cells were treated with pp242 (A) or transfected with RICTOR siRNA (B) and (C) to inhibit mTORC2 activity and then irradiated by 30 mJ/cm² of UVB. Scrambled siRNA was used as a negative control. The levels of Rictor, phosphor-Akt (Ser473 and Thr308), phospho-IKKs, and nuclear p65 were measured by Western blotting (A and B) and confocal microscopic analysis detected p65 translocation (C). One representative experiment is shown from 3 experiments that yielded similar results.

Figure 5. A schematic model showing the underlying mechanism of skin aging

UVB or age increases the activity of mTORC2 that phosphorylates Akt. In turn, Akt phosphorylates IKKα at Thr23, leading to the translocation and phosphorylation of p65 during skin aging.