Smad7 Is Highly Expressed in Human Degenerative Discs and Participates in IL-1β-Induced Apoptosis of Rat AF Cells via the Mitochondria Pathway

Background Abnormal Smad7 expression can lead to apoptosis in different cell types. Previously, we found high expression of Smad7 in rat degenerative discs. However, the exact role of Smad7 in the apoptosis of disc cells and the possible underlying mechanism remain unclear. Methods Degenerative and nondegenerative human lumbar intervertebral discs were collected from patients during operation. The expressions of SMAD7 mRNA and protein in the different components of these discs were measured with real-time PCR and Western blotting, respectively. Annulus fibrosus (AF) cells were isolated and cultivated from the discs of young healthy rats. Smad7 in the AF cells was overexpressed with adenovirus and knocked down with siRNA. IL-1β was used to induce apoptosis in the AF cells. Loss-and-gain cell function experiments were performed to show the effect of Smad7 on the apoptosis of AF cells. The function recovery experiments were performed to verify whether Smad7 regulates the apoptosis of AF cells through the mitochondria-mediated pathway. Results Both the mRNA and protein expressions of Smad7 were significantly higher in the different components of human degenerative discs than in those of the nondegenerative discs. IL-1β stimulated apoptosis while upregulating the Smad7 expression in the AF cells in vitro. Overexpression of Smad7 in AF cells exaggerated the IL-1β-induced apoptosis in the cells while knockdown of Smad7 expression suppressed this apoptosis. With the exaggerated apoptosis in the AF cells with Smad7 overexpression, both active cleaved caspase-3 and cleaved caspase-9, the ratio of Bax/Bcl-2, and Cyt-c increased significantly. However, the inhibitor of caspase-9, Z-LEHD-FMK, significantly diminished the apoptosis in these cells. Conclusion Smad7 is highly expressed in human degenerative discs and participates in IL-1β-induced apoptosis of rat AF cells via the mitochondria pathway. Smad7 may be a potential target for the prevention and treatment of degenerative disc disease.


Introduction
Intervertebral disc degeneration disease (IVDD) is one of the most common causes of disability in aging people and a decline in the quality of their life [1][2][3][4]. However, the pathogenesis of IVDD is not fully understood. A decrease in the number of live cells in the disc and reduced intracellular matrix with altered components are the pathological basis of disc degeneration [2,5]. Increased apoptosis of disc cells is the primary reason for the decrease in the number of live cells [2,5,6]. Excessive apoptosis could also lead to a decrease in cell density and an increase in intracellular matrix catabolism, resulting in disc degeneration [7][8][9]. Thus, suppressing the excessive apoptosis of disc cells is believed to be a potential way to delay disc degeneration.
Smad7 is a key member closely related to TGF-β functions. It binds to the TGF-β receptor, inhibits the phosphorylation of Receptor-Smad complex, and blocks the signal transduction of TGF-β. Meanwhile, TGF-β could induce Smad7 expression through the activation of Receptor-Smads, thus forming a positive feedback regulation. Activated Receptor-Smads could also upregulate Smad7 expression through the activation of Smad7 promoter, forming another regulatory feedback loop [10]. By blocking TGF-β signaling pathways, Smad7 inhibited the synthesis of collagen and proteoglycans. It has been reported that adenovirus with Smad7 cDNA transfected into hepatic stellate cells or renal interstitial fibroblasts inhibited the synthesis of collagen and proteoglycan and stopped the process of fibrosis [11,12]. Our previous study also has demonstrated that Smad7 plays key roles in disc degeneration. Smad7 was found to be highly expressed in degenerated intervertebral discs in rats, and Smad7 expression was closely associated with the metabolism of the intracellular matrix [13]. It has been reported that Smad7 promoted apoptosis by blocking the NF-κB/p65 pathway in glomerular podocytes [14]. However, the exact role of Smad7 in the apoptosis of disc cells and the possible underlying mechanism remain unclear.
IL-1β is significantly increased in degenerative intervertebral discs (IVDs) [15]. An increasing number of studies have proved that IL-1β is involved in the inflammatory response in the process of intervertebral disc degeneration. Exposure of human AF cells to IL-1β can significantly increase apoptosis [16,17]. Our previous study also con-firmed that IL-1β is involved in IVDD and IL-1β enhances the effect of serum deprivation on rat AF cell apoptosis [18]. IL-1β was reported to induce the expression of Smad7 in chondrocyte [19]. But it is still unclear whether Smad7 participates in IL-1β-induced AF cell apoptosis.
Based on the above-mentioned findings, we continue our study on the role of Smad7 in disc degeneration. In this study, we further investigated the expression of SMAD7 in human degenerative discs and clarified the role of Smad7 in the IL-1β-induced apoptosis of rat AF cells. The underlying signaling pathways of apoptosis are also explored. We hope our results will provide a new idea for the prevention and treatment of IVDD.  Figure 1: The expression of Smad7 in human degenerative and non-degenerative intervertebral discs. (a) qRT-PCR assays to detect the mRNA expression of SMAD7 in human degenerative and nondegenerative intervertebral discs. (b) Western blotting assays to detect the protein expression of SMAD7 in human degenerative and nondegenerative intervertebral discs. p values less than 0.05 were considered statistically significant. NP: nucleus pulposus; AF: annulus fibrosus; EP: endplate; * * * p < 0:001, * * p < 0:01, and * p < 0:05. All experiments were performed at least three times. 2 Oxidative Medicine and Cellular Longevity criteria on MRI, 4 discs were grade I, 10 were grade II, 11 were grade III, 11 were grade IV, and 9 were grade V. These 45 discs were divided into 2 groups: nondegenerative (grades I-II) and degenerative (grades III-V). The annulus fibrosus, nucleus pulposus, and endplate tissues of each disc were carefully separated in the operation table and appropriately stored for the following experiments. Human AF tissue collection and animal experiments were approved by the Ethics Committee of our university hospital (No. XHECD2015107). All experiments involving human specimens followed the Helsinki declaration.

Materials and Methods
2.2. Isolation and Culture of Rat AF Cells. The whole lumbar spine segments were dissected from eight healthy 3-monthold male Sprague-Dawley rats after sacrifice by intraperitoneal injection of over-dosed Sumianxin. L1/2, L2/3, L3/4, L4/5, and L5/6 discs were separated. AF cells were isolated and cultured according to our previous method [18,20]. First-passage cells     7 Oxidative Medicine and Cellular Longevity maintained in a monolayer were used throughout the experiments [21]. The isolated primary AF cells were verified by the characteristics of their cell morphology [22].

Adenovirus-Mediated Gene
Transfer into AF Cells. The adenovirus vector with Smad7 gene overexpression was constructed by the manufacturer (Gene Pharma, China). On the day of transfection, AF cells were seeded into 24-well culture dishes at a density of 1 × 10 5 cells/well. When they reached 50% confluence, cells were transfected with the Smad7 overexpression adenovirus or negative control adenovirus at a multiplicity of infection of 100, and cells without transfection were used as control. The efficacies of transfection were determined by western blotting and real-time PCR.
Quantitative densitometric values of each protein of interest were normalized to GAPDH.

Nuclear Staining Using Hoechst 33258.
Three groups of AF cells were seeded in 96-well culture plates and added with Hoechst 33258 dye solution (Beyotime, C0003) followed by removing the culture media at 12, 24, and 48 h, respectively. The procedures were done as previously described [21]. After washing with PBS twice, AF cells were added with 0.5 ml 4% formaldehyde solution and fixed at RT for 10 min. Then, AF cells were washed with PBS for 2-4 times and incubated with Hoechst 33258 solution for 20-60 min in the dark at RT. Cells were observed under a fluorescence microscope under UV light followed by washing with PBS and adding with antifade reagent.

Cell
Apoptosis Assay Using Flow Cytometry. AF cells overexpressing Smad7 were first collected for apoptosis assay using flow cytometry at 12, 24, and 48 h, respectively. And then, we used LEHD-FMK to confirm the involvement of the mitochondria-mediated pathway in the apoptosis of rat AF cells after 48 h of Smad7 overexpression. The experiments were performed according to the manual of Annexin V-FITC Apoptosis Detection Kit (BD Pharmingen TM) [7]. In brief, AF cells were collected by centrifuge at 1000 g for 5 min followed by wash with PBS and digestion using Trypsin-EDTA. After resuspension in PBS, cell numbers were counted and 5-10 × 10 4 cells were aliquoted for staining with Annexin V-FITC solutions for 10 min in the dark at RT. Propidium iodide (PI) was then added to the AF cells. All the stained cells were analyzed using a flow cytometry platform for Annexin V-FITC and PI. 2.11. Statistical Analysis. All data were analyzed using SPSS11.5 statistical software. Comparisons of data between groups were analyzed using one-way ANOVA, Kruskal-Wallis H-test, or independent-samples t-test according to the distribution of data and homogeneity of variance. p values less than 0.05 were considered statistically significant.

Smad7 Is Highly Expressed in the Human Degenerative
Discs. The expression of Smad7 in degenerative NP, EP, and AF was significantly higher than that in the nondegenerative counterpart (Figure 1(a)). The Smad7 protein expressions showed similar variation trends as compared with PCR analysis (Figure 1(b)). But Smad7 expressions were much higher in the AF than in NP and EP, which indicated that Smad7 might have a more important role in the AF than in the other components of the disc.

IL-1β Induces Apoptosis in Rat AF Cells through Its
Regulations on Smad7. It has been proved that IL-1β induces apoptosis in AF cells [18,24,25]. We investigated whether Smad7 was involved in the IL-1β-induced apoptosis of AF cells. With the stimulation of IL-1β, the apoptosis-related proteins, such as cleaved caspase-9 and cleaved caspase-3, significantly increased in the AF cells (Figure 2(a)). Smad7 mRNA (Figure 2(b)) and protein (Figure 2(c)) expressions were also upregulated with time in these AF cells. Successful knockdown of Smad7 in AF cells was verified at both the gene (Figure 3(a)) and protein (Figure 3(b)) levels.
Overexpression of Smad7 in AF cells with adenovirus infection was also successful (Figures 4(a) and 4(b)). On the contrary, with the overexpression of Smad7 in AF cells, the IL-1β-induced apoptosis was greatly exaggerated (Figure 4(c)). More chromosome condensation and nuclear fragmentation under Hoechst 33258 staining were seen in the cells (Figure 4(d)). Taken together, these results indicated that IL-1β-induced apoptosis in rat AF cells through its regulations on Smad7. pathway and the intrinsic (also called mitochondrial) pathway [26]. The extrinsic pathway requires external stimulation, and this occurs via a death receptor (DR). The intrinsic pathway involves a wide array of stimuli that are sensed intracellularly, including cytokine deprivation, DNA damage, and endoplasmic reticulum (ER) stress [27]. These diverse apoptotic stresses converge to trigger one crucial event-mitochondrial outer membrane permeabilization (MOMP) [28]. It is reported that TGF-β1-mediated apoptosis was associated with Smad7-dependent mitochondrial Bcl-2 expression [29] and Smad7 increased TGF-β-mediated apoptosis in Mv1Lu cells [30]. These studies indicated that Smad7 might lead to cell apoptosis via the mitochondria signaling pathway.
To investigate whether Smad7 participates in IL-1βinduced apoptosis of rat AF cells via the mitochondria signaling pathway, we measured the protein expression of different biomarkers of the mitochondria-mediated apoptosis. Western blot analysis revealed that the protein level of Bax in rat AF cells overexpressing Smad7 significantly increased, while the levels of both Bcl-2 and Bcl-XL significantly decreased, which resulted in even greater increase in the ratio of Bax/Bcl-2 ( Figure 5(a)). Meanwhile, the expression of both active cleaved caspase-3 and cleaved caspase-9 also increased significantly in the AF cells with Smad7 overexpression. However, the protein expressions of procaspase-3 and procaspase-9 did not show any significant changes ( Figure 5(a)). In addition, the expression of Cyt-c also increased significantly ( Figure 5(b)). These results indicated that the mitochondrial pathway is likely involved in the Smad7-mediated apoptosis induced by IL-1β. Smad7 may promote Bax expression by inhibiting Bcl-2 and Bcl-XL expression and activating the mitochondria-mediated apoptosis pathway.
To further confirm the involvement of the mitochondria pathway in the Smad7-mediated apoptosis, AF cells with overexpression of Smad7 were treated with Z-LEHD-FMK, an inhibitor of caspase-9. After treatment with Z-LEHD-FMK, the protein expression of active cleaved-caspase-9 in Ad-Smad7-infected cells was downregulated by one-fold ( Figure 5(c)), which is consistent with results reported in previous studies [31][32][33]. The apoptosis percentage was significantly lower in Ad-Smad7-infected cells treated with Z-LEHD-FMK than in cells without Z-LEHD-FMK treatment; however, it was still higher than that in Con and Ad-Con cells ( Figure 5(c)). This suggested that Z-LEHD-FMK could not completely inhibit the Smad7-mediated apoptosis in AF cells. Immunofluorescence assay for the detection of protein expression of cleaved caspase-9 showed that Ad-Smad7 increased the level of cleaved caspase-9, while Z-LEHD-FMK weakened this effect of Ad-Smad7 ( Figure 5(d)). These results further provided evidence that Smad7 participates in IL-1β-induced apoptosis of rat AF cells via the mitochondria signaling pathway.

Smad7
Regulates the IL-1β-Induced Apoptosis in AF Cells by Interfering with the Formation of Smad2/Smad3/ Smad4 Complex. With the stimulation of IL-1β, the protein expressions of p-Smad2, p-Smad3, and Smad4 significantly decreased in the AF cells (Figure 6(a)). However, in cells with a successful Smad7 knockdown, the expression of p-Smad2, p-Smad3, and Smad4 was not affected by IL-1β treatment. Furthermore, in the cells with Smad7 knockdown, the nuclear translocation of p-Smad2/p-Smad3 significantly increased (Figure 6(b)). These results demonstrated that Smad7 could regulate the IL-1β-induced apoptosis in AF cells by interfering with the formation of Smad2/ Smad3/Smad4 complex.

Discussion
Smad7 is a negative regulatory protein involved in the signal transduction of TGF-β pathway. It blocks signal transduction and inhibits the transcription of TGF-β gene. In our previous study, we observed that Smad7 was highly expressed in degenerative intervertebral discs of rats and that this was closely related to the catabolism and apoptosis of disc cells [13]. However, the underlying mechanisms remained unknown.
A clue indicating a potential role of Smad7 in IVDD came from the studies showing that TGF-β1 could promote proteoglycan synthesis and cell proliferation of NP and AF cells cultured in vitro and in vivo [7,[34][35][36][37]. As a downstream molecule of TGF-β1, exploring the role and mechanism of Smad7 in intervertebral disc degeneration has grabbed our interest. In this study, we found that Smad7 was highly expressed in human degenerative discs. Combined with the finding in our previous animal study that the expression of Smad7 significantly increased in the degenerative discs of rats, we verified that Smad7 should be involved in the pathophysiological process of intervertebral disc degeneration.
It has been proved that IL-1β has important roles in the pathogenesis of disc degeneration by stimulating the apoptosis in disc cells [17,18]. In this study, we verified that IL-1β could stimulate apoptosis in the AF cells isolated and cultured from rats and found increased Smad7 expression in the cells that along with this IL-1β-induced apoptosis. In order to investigate the role of Smad7 in the apoptosis of AF cells and the underlying signaling pathway, Smad7 was overexpressed or knocked down in the AF cells under IL-1β stimulation. Overexpression of Smad7 in the AF cells exaggerated the IL-1β-induced apoptosis, while knockdown of Smad7 in the cells significantly diminished the IL-1β-induced apoptosis. Meanwhile, we found that with the increase of apoptosis in the cells with Smad7 overexpression, the proteins in the mitochondrial mediated apoptosis pathway, such as active cleaved caspase-3 and cleaved caspase-9, the ratio of Bax/Bcl-2, and Cyt-c increased significantly. However, the inhibitor of caspase-9, Z-LEHD-FMK, significantly diminished the apoptosis in these cells. Smad7 regulated the apoptosis of the AF cells through the mitochondrial signaling pathway. Our results also demonstrated that Z-LEHD-FMK did not completely block the activation of caspase-9 in the AF cells; it did not completely offset the increased apoptosis in the AF cells with Smad7 overexpression. The reasons for this incompetency of Z-LEHD-FMK might be as follows: (1) Z-LEHD-FMK did not completely bind to caspase-9 in the AF cells, resulting in an incomplete inhibition of apoptosis in the cells with Smad7-overexpression. (2) Caspase-9 in the middle of the apoptotic cascade is the upstream molecule of caspases 3, 6, and 7 [28,38,39]. Smad7 activated the downstream molecules of caspase-9 through other means. (3) Smad7 did not completely activate the apoptosis through the mitochondrial pathway, and hence, other pathways might be involved.
High expression of Smad7 was reported in chondrocytes under IL-1β stimulation [19]. Although IL-1β is proved to be able to induce disc cell apoptosis and disc degeneration, the expression of Smad7 in the AF cells under IL-1β stimulation has never been described. In our study, we found that IL-1β led to an increase in Smad7 expression and a decrease in p-Smad2, p-Smad3, and Smad4 expressions in the AF cells. After successful Smad7 knockdown in the AF cells, stimulation of IL-1β did not change the expressions of p-Smad2, p-Smad3, and Smad4. Combined with previous data and studies [25,40,41], we believed that Smad7 should be involved in the apoptosis of AF cells by interfering with the formation of the Smad2/Smad3/Smad4 complex and regulate the apoptosis through the mitochondrial signaling pathway.
In conclusion, our study demonstrated that Smad7 was highly expressed in human degenerative intervertebral discs and that Smad7 regulated apoptosis of the AF cells mainly through the mitochondria signaling pathway. Smad7 might be a potential target for the prevention and treatment of IVDD.

Data Availability
Data can be obtained from the corresponding authors if required.