Akt inhibition up-regulates MMP1 through a CCN2-dependent pathway in human dermal fibroblasts

Abstract

Akt is a key signalling molecule that was found to be down-regulated in chronic wounds. Akt blockade has dual antifibrotic effects in human dermal fibroblasts, by up-regulating matrix metalloproteinase 1 (MMP1) and down-regulating collagen gene expression (J Invest Dermatol 2008: 128: 1906). The aim of this study was to gain additional insights into the mechanism of MMP1 up-regulation following Akt blockade. As previous studies showed that CCN2 can be a positive regulator of MMP1, we examined the effects of Akt inhibition on CCN2 expression. Akt blockade using a specific pharmacological inhibitor and Akt siRNA resulted in a significant up-regulation of CCN2, which correlated with the increase in MMP1. The MMP1 up-regulation following Akt blockade was partially suppressed by CCN2 siRNA, suggesting that CCN2 is contributing to this effect. Additional experiments showed that CCN2 induces phosphorylation of ERK1/2, Ets1 and c-Jun. Consistent with the stimulatory role of ERK1/2/Ets1 in the expression of MMP1, the ERK1/2 inhibitor UO126 prevented the phosphorylation of ERK1/2 and Ets1 and completely abrogated the induction of MMP1 after CCN2 overexpression, while having no effect on c-Jun activation. Taken together these results establish CCN2 as a key regulator of MMP1 induction via activation of the ERK1/2/Ets1 pathway. Down-regulation of Akt signalling leads to inappropriate activation of the CCN2/MMP1 pathway that may contribute to the pathogenesis of chronic wounds. Coordinate expression of CCN2, Akt and MMP1 could be important for normal wound healing to occur. Thus, targeting these specific proteins may represent a promising approach to the therapy of dysregulated wound healing.

Figure 1

CCN2 is a positive regulator of matrix metalloproteinase 1 (MMP1). Equal numbers of human dermal fibroblasts were plated and grown to confluence then serum-starved overnight. Cells were then treated with either CCN2 over-expressing or control (G0) adenovirus in (a) and with recombinant human CCN2 in (b). Western blot from conditioned media collected 72 h latter shows that compared to control, cells treated with CCN2 secreted more MMP1 and less collagen type I, in a dose-dependent manner. Bar graphs in (b) represent means ± SEM of three independent experiments in two different cell lines, with *P ≤ 0.05; **P ≤ 0.01.

Figure 2

The effects of Akt inhibition on basal and TGFβ-induced CCN2 levels. (a) Dose-dependent increase in CCN2 with Akt inhibitor VIII. Dermal fibroblasts were grown to confluence and serum-starved overnight then treated with either Akt inhibitor VIII (2.5, 5, 10, 15 and 20 µm) or DMSO for 2 days. Cells were scraped and the levels of CCN2 were analysed by Western blot. (b) Time-dependent up-regulation of CCN2 mRNA levels after Akt inhibition. Dermal fibroblasts were treated with 20 µm of Akt inhibitor VIII for the indicated times and then the mRNA levels were analysed by qRT–PCR. mRNA values were normalized relative to control, DMSO-treated cells (arbitrarily set as 1) and means ± SEM of four independent experiments in three different cell lines are shown. *P < 0.05; **P < 0.01 versus control, vehicle-treated cells. (c) Representative Western blot showing the effect of Akt 1, 2 and 3 siRNA on CCN2 protein levels. The levels of total Akt, CCN2 and β-actin were analysed by Western blot. Representative data from three different experiments are shown with quantitative analysis of the results at right: the bar graph shows means ± SEM with values normalized to control, scrambled siRNA-treated cells. *P < 0.05; **P < 0.01. (d) Equal numbers of dermal fibroblasts were grown to confluence and serum-starved overnight then treated with increasing doses of Akt inhibitor VIII for 72 h. A representative Western blot of conditioned media showing the levels of CCN2 and matrix metalloproteinase 1 (MMP1) is shown. The experiments were repeated at least three times in two different cell lines. (e) Confluent dermal fibroblasts were serum-starved overnight and pretreated with 20 µm of Akt inhibitor VIII for 1 h, followed by TGFβ stimulation for 72 h. Cells were collected and the levels of CCN2 and MMP1 were analysed by Western blot or qRT–PCR for MMP1 in (f). β-actin was used as a control. Experiments were repeated at least three times in two different cell lines, and a representative gel is shown. *P < 0.05; **P < 0.01.

Figure 3

Increased CCN2 in response to Akt blockade contributes to matrix metalloproteinase 1 (MMP1) up-regulation. (a) Effects of adenoviral-mediated CCN2 overexpression and Akt inhibitor VIII on MMP1 levels. Equal numbers of dermal fibroblasts were grown to confluence, serum-starved overnight and then treated with 20 µm of Akt inhibitor VIII or transduced with adenovirus overexpressing CCN2 for 72 h. DMSO and G0 adenovirus were used as control. Western blot analysis of conditioned media shows the increase in MMP1 secretion of cells with different levels of CCN2 up-regulation. A representative blot is presented and the bar graph shows means ± SEM of three independent experiments with values normalized to control, vehicle-treated cells, *P < 0.05, **P < 0.01. (b) Effects of siRNA-mediated CCN2 blockade on Akt inhibitor VIII-mediated MMP1 up-regulation. Equal numbers of dermal fibroblasts were plated and treated with adenovirus overexpressing CCN2siRNA in 10% fetal bovine serum. Twenty-four hours later media was changed to serum free media and cells were treated with 2.5 ng/ml of TGFβ or 20 µm of Akt inhibitor VIII. Conditioned media collected after 72 h was analysed by Western blot for secreted CCN2 and MMP1. Representative blots are shown with quantitative analysis of three different experiments in three different cell lines on the right panel. The values were normalized to control, vehicle-treated cells, *P < 0.05, **P < 0.01.

Figure 4

CCN2 induces matrix metalloproteinase 1 (MMP1) gene expression via an ERK1/2/Ets1-dependent mechanism. (a) Adenovirus-mediated transfer of dominant-negative Akt₁ enhances the basal and TGFβ-induced phosphorylation of Erk1/2 in human dermal fibroblasts in a time-dependent manner. Dermal fibroblasts were transduced with either AdDnAkt₁ or control virus (LacZ) at MOI 50, as described. Cells were treated after 48 h with 1 ng/ml of TGFβ for the indicated time points and then collected. The levels of phospho-ERK1/2 and total ERK1/2 were analysed by Western blot. Experiments were repeated twice in two different cell lines. (b) Time-dependent induction of Ets1 and ERK1/2 phosphorylation by CCN2. Cells were treated with recombinant human CCN2 for the indicated time points and the levels of P-Ets1, Ets1, P-ERK1/2 and ERK1/2 were analysed by Western blot. Experiments were repeated twice in two different cell lines. (c) Effects of UO126 on cells overexpressing CCN2. Dermal fibroblasts were pretreated with UO126 for an hour and then transduced with either AdCCN2 or control virus (LacZ) as described. The levels of CCN2, MMP1, P-Ets1, P-ERK1/2 and P-c-Jun were analysed by Western blot 48 h after transduction. β-actin was used as a control for equal loading. Representative gels are shown. Experiments were repeated twice in two different cell lines.

Figure 5

The effects of Akt activation status on matrix metalloproteinase 1 (MMP1) gene expression. In the presence of pharmacological inhibitors, signalling through Akt is blocked resulting in an increase in CCN2 protein levels, which in turn activates ERK1/2/Ets1 signalling leading to transcriptional activation of the MMP1 promoter. Conversely, activated Akt downstream of TGFβ or other stimuli may be a negative regulator of MMP1 gene expression via additional unknown mechanisms.