The role of integrin alpha(v)beta (8) in neonatal hypoxic-ischemic brain injury

Abstract

Integrin alpha(v)beta(8) plays an important role in cerebral vascular development. It has been proven that alpha(v)beta(8) is a key factor for transforming growth factor-beta1 (TGF-beta1) activation in epithelial cells. However, it is not clear whether alpha(v)beta(8) can activate TGF-beta1 and play a role in protection during neonatal hypoxic-ischemic brain injury. In this study, we investigated the relationship between alpha(v)beta(8) and TGF-beta1 activation, and thus the effects of TGF-beta1 activation in the protection of neurons after hypoxia-ischemia (HI). Astrocytes and neurons from rat brains were cultured and then subjected to oxygen-glucose deprivation to generate HI model in vitro. beta(8) expression was determined using immunocytochemistry, western blot, and reverse-transcriptase polymerase chain reaction. TGF-beta1 activation was determined by TGF-beta bioassay in a tested cell (astrocyte) and a reporter cell co-culture system. The pro-apoptotic protein, cleaved caspase-3, and the anti-apoptotic protein, Bcl-2 and Bcl-xL, were detected using western blot. Cellular apoptosis was detected with TUNEL. We found that beta(8) expression was stronger in astrocytes than that in neurons under normoxia. HI resulted in a rapid and persistent increase of beta(8) expression in astrocytes, but only in a slight and transient increase in neurons. Astrocytes beta(8) could induce TGF-beta1 leading to upregulation of Bcl-2 and Bcl-xL, and thus attenuated neuronal apoptosis. The present findings suggest that beta(8) protecting the brain against neonatal HI injury through TGF-beta1 signaling pathway, which may have implications for the treatment of HI brain injury.

Fig. 1

Immunoreactivity of β₈ in normoxic cultured astrocytes and neurons (N = 5). β₈ was strongly expressed in astrocytes (b) but with a weak expression in neurons (d). The positive immunoreactivity was mainly located in the membrane, cytoplasm, and neurites of the cells (b, d). There was no positive immunoreactivity in negative controls (a, c). Arrows show the positive staining cells. × 400

Fig. 2

The effects of HI on β₈ expression in cultured astrocytes and neurons. β₈ mRNA expression was increased at 6 h after reoxygenation, peaked at 1 day, then slowly decreased but still maintained at high level at 7 days in astrocytes compared with controls (a). HI also resulted in an increase of β₈ mRNA expression in neurons at 6 h, but declined at 12 h and returned to baseline within 2 days after reoxygenation (b). Analysis showed that the β₈ protein was increased at 6 h, and maintained for at least 7 days after reoxygenation in astrocytes (c), but slightly and transiently increased in neurons at 6 h after reoxygenation and quickly returned to baseline within 1 day (d). Quantification of β₈ expression in astrocytes and neurons, respectively, in HI groups and normoxic controls (e, f). Results were normalized to controls and represented as mean ± SD. For each column, N = 4, * P < 0.05, ** P < 0.01 versus control

Fig. 3

Effects of RNAi on β₈ expression in astrocyte cultures. Real-time RT-PCR and western blot analysis were used to detect the expression of β₈ in cultured astrocytes after infection. The relative amounts of β₈ mRNA in astrocytes treated with β₈ siRNA (β₈ RNAi) or control siRNA (CTRL) virus were determined by using the standard-curve method. The highest inhibition rate was around 80% at 2 days (a). Western blot analysis showed β₈ protein expression was significantly inhibited in β₈ knockdown astrocytes with a maximal inhibition rate around 84% at 2 days (b, c). Results were normalized to the non-infected astrocytes (Non) and represented as mean ± SD. For each column, N = 3, ** P < 0.01 versus Non & CTRL

Fig. 4

Astrocytes β₈ mediates activation of TGF-β1 after HI following reoxygenation. TGF- β 1 activation was increased significantly after co-cultured with ${\beta }_8^{+/+}$ astrocytes under normoxic condition (a). TGF- β 1 activation was increased after reoxygenation compared with the normoxic controls (b). TGF- β 1 activation was significantly lower in ${\beta }_8^{-/-}$ astrocytes than that in ${\beta }_8^{+/+}$ astrocytes (c). TGF- β 1 activation was significantly increased in ${\beta }_8^{+/+}$ astrocytes but not in ${\beta }_8^{-/-}$ astrocytes after reoxygenation (d). TGF- β 1 activation was inhibited while treated with GM6001, especially by β ₈ knockdown ( ${\beta }_8^{-/-}$) (e). For each column, N = 5, ** P < 0.01 versus anti-TGF β & TMLC alone (a); ** P < 0.01 versus ${\beta }_8^{+/+}$, P < 0.05, ^##P < 0.01 versus control (b); ** P < 0.01 versus ${\beta }_8^{-/-}$ (c); **P < 0.01 versus control (d); ** P < 0.01 versus GM6001, *P < 0.05 versus GM6001 ^##P < 0.01versus ${\beta }_8^{-/-}$ (e)

Fig. 5

Integrin β ₈/TGF- β 1 signaling pathway protected neurons from apoptosis after OGD. TUNEL positive cells expressed stronger in neurons at 1 day after reoxygenation (Ac, R1d) compared with the neurons in normoxic controls (Ab, con) and in negative controls (Aa). The expression of TUNEL positive cells were significantly decreased while co-cultured with ${\beta }_8^{+/+}$ astrocytes (Ad, Non). However, after blocking TGF-β 1 or blocking β ₈, the number of TUNEL positive cells was increased (Ae, Af). Western blot analysis showed that CC3 expression was significantly increased at 1 day after reoxygenation in neurons cultured alone (B, R1d) compared with the normoxic controls. CC3 expression was significantly decreased while co-cultured with ${\beta }_8^{+/+}$ astrocytes (B, Non), but was rescued while blocking TGF-β (B, anti-TGF β) or β ₈ (B, β₈ siRNA). Quantification of CC3 expression in neurons cultured alone and neurons co-cultured with astrocytes (C). Results were normalized to normoxic controls and represented as mean ± SD. For each column, N = 4, **P < 0.01 versus con & Non, ^## P < 0.01 versus Non

Fig. 6

The integrin β₈/TGF-β1 signaling pathway protected neurons from apoptosis by upregulating anti-apoptotic protein expression after HI. Western blot analysis showed that Bcl-2 expression was decreased in neurons cultured alone at 1 day after reoxygenation (a, R1d) compared with controls (a, con). However, Bcl-2 was significantly increased while co-cultured with ${\beta }_8^{+/+}$ astrocytes (a, Non) but could be partially inhibited by either TGF-β antibody (a, anti-TGF β) or β₈ knockdown (a, β₈ siRNA). Bcl-xL did not show significant changes in neurons cultured alone at 1 day after reoxygenation (b, R1d) compared with controls (b, con). Bcl-xL was significantly increased while co-cultured with ${\beta }_8^{+/+}$ astrocytes (b, Non) but could be reduced by TGF-β antibody (b, anti-TGF β) or β₈ knockdown (b, β8 siRNA). Quantification of Bcl-2 and Bcl-xL expression (c, d). Data were normalized to controls and represented as mean ± SD. For each column, N = 5, ** P < 0.01, ^## P < 0.01 versus Non (c); **P < 0.01, * P < 0.05 versus Non (d)