A hyperglycemic microenvironment induced prolonged ER stress responses in the human gingival epithelium
Gingival epithelium ER homeostasis play a key role in defending against exogenous infection. An increasing number of studies has indicated that in cells are subjected to prolonged ER stress, the intracellular calcium concentration increases and disrupts ER homeostasis [25]. To clarify how a high-glucose microenvironment impairs HGEC function, flow cytometry analysis was performed to examine cytosolic Ca2+ stained with Fluo-4AM. Intriguingly, a high-glucose microenvironment induced a significantly increase in the intracellular calcium ion concentration in HGECs (Fig. 2A, 2B), suggesting that a high-glucose microenvironment induces prolonged ER stress responses and perturbs ER homeostasis in HGECs.
To investigate the mechanisms responsible for prolonged ER stress responses in periodontitis under diabetic conditions, we sought to understand the role of IRE1α signaling within HGECs under hyperglycemia. IRE1α is a bifunctional enzyme that processes an RNase domain and a kinase domain. We detected IRE1α activity in HGECs; the treatment of HGECs with glucose at a high concentration (25 mM) for 48 h diminished the expression of IRE1α and increased the levels of phosphorylated IRE1α (p-IRE1α) compared to those in HGECs treated with 5.5 mM glucose (Fig. 2C-2E), indicating that the expression and activity of IRE1α are defective in HGECs cultured in high-glucose conditions. IRE1α plays an important role in maintaining ER homeostasis by initiating the unconventional splicing of XBP1 mRNA to create a translational frame shift in XBP1 mRNA. This produces a potent transcription factor, XBP1-s, which regulates the expression of genes with functions in ER protein folding and trafficking and ER-associated degradation to preserve ER homeostasis [26]. We used real-time polymerase chain reaction (PCR) to measure the mRNA expression levels of ER stress markers in HGECs treated with 5.5 mM or 25 mM glucose for 48 h. The level of un-spliced XBP1 (XBP1-u) mRNA was increased in HGECs treated with 25 mM glucose compared with HGECs treated with 5.5 mM glucose, but a corresponding increase in XBP1-s was absent, indicating a defect in the processing of XBP1-u to XBP1-s by IRE1α RNase activity despite the phosphorylation of IRE1α (Fig. 2E). To determine whether a progressive decline in XBP1-s in hyperglycemia also affects the regulation of UPR target gene expression, we studied the effect of glucose on glucose regulated protein 78 (GRP78) activation by treating HGECs with glucose at one of two concentrations, 5.5 mM and 25 mM. The treatment of HGECs with glucose at a high concentration (25 mM) for 48 h decreased the level of GRP78 compared to that upon treatment with 5.5 mM glucose (Fig. 2C-2E), indicating the impaired resolution of ER stress under hyperglycemia. In summary, these results demonstrated that the IRE1α signaling pathway is inhibited in HGECs under high-glucose conditions, indicated that failure of the ER stress response and UPR activation completely overwhelm cytoprotective mechanisms in response to hyperglycemia, resulting in prolonged or severe ER stress.
Subsequently, to mimic the inflammatory environment, HGECs were stimulated with Porphyromonas gingivalis lipopolysaccharide (LPS). Intriguingly, the results of RT-qPCR and western blotting also indicated that IRE1α, XBP1-s and GRP78 levels remained high, whereas XBP1-u levels remained low after HGECs were stimulated with 1 µg/mL P. gingivalis LPS (Fig. 2C-2E). Through its RNase activity, activated IRE1α directly splices XBP1 mRNA to produce XBP1-s, which then upregulates the expression of genes downstream of IRE1α such as GRP78, thereby reducing misfolded or unfolded proteins in the ER. Our results suggest that P. gingivalis LPS-stimulated HGECs may be responsible for the initiation of adaptive ER stress to restore homeostasis, but in the presence of hyperglycemia, cellular stress exceeds the capacity of the UPR, and the UPR machinery is damaged. This notion also reflects the poorer control of periodontal inflammation by conventional periodontal treatments in patients with periodontitis with diabetes mellitus compared with periodontitis patients.
Next, to analyze the expression of IRE1α and GRP78 in the human periodontium, we performed immunohistochemical staining of the human gingival epithelium for IRE1α and GRP78 (n = 14). IRE1α and GRP78 immunoreactivity in the human gingival epithelium was significantly downregulated in the DP group and significantly upregulated in the P group compared with the healthy group (Fig. 2F-2I). Pearson’s correlation analysis was conducted and revealed a positive relationship between IRE1α and GRP78 expression within tissues of the human gingival epithelium (Fig. 2J-2L). These results demonstrate impairment of the IRE1α signaling pathway and prolonged ER stress in the gingival epithelium in periodontitis with diabetes mellitus and suggest that prolonged and exaggerated ER stress is related to the presence of hyperglycemia.
Decreased SERPINH1 expression in the gingival epithelium in periodontitis with diabetes mellitus
To better understand the mechanism by which severe inflammation in the periodontium occurs under diabetic conditions, we subjected the gingival epithelium of mice with periodontitis with diabetes mellitus and mice with periodontitis to RNA sequencing (RNA-seq) data analysis. We analyzed ER-related genes and identified differentially expressed genes based on biological process. Among the ER-related genes, SERPINH1, ATP2A1, CASQ1, CYP2E1, STBD1, RBL21 and TMEM38A showed significantly different expression between the DP group and P group (Fig. 3B, 3C). RT-qPCR analysis further confirmed that SERPINH1 mRNA expression levels were significantly decreased in HGECs under high-glucose (25 mM) conditions compared with those without high-glucose treatment (Fig. 3A). The decrease in the SERPINH1 level in HGECs under high-glucose conditions was further confirmed by western blot analysis (Fig. 3D, 3H).
We further evaluated the expression level of SERPINH1 in the human gingival epithelium (n = 14) using immunohistochemistry. A sharp contrast in SERPINH1 tissue staining was observed between the DP and P groups. According to the results of immunohistochemical staining for SERPINH1, the expression of SERPINH1 in the human gingival epithelium was lower in the DP group and higher in the P group than in the healthy group (Fig. 3I, 3J). Taken together, these data suggest that decreased SERPINH1 protein expression is associated with diabetes. Furthermore, Pearson’s correlation analysis was conducted, and a positive relationship between SERPINH1 and IRE1α expression within human gingival epithelial tissues was found (Fig. 3E-3G), which indicates that significant downregulation of SERPINH1 in a hyperglycemic microenvironment may lead to prolonged or severe ER stress.
Silencing of SERPINH1 significantly prolonged ER stress responses and initiated an abnormal inflammatory response in HGECs
To ascertain the effect of SERPINH1 inhibition of ER stress responses and the inflammatory response, we transfected SERPINH1 siRNA into HGECs. RT-qPCR and western blot analysis confirmed the good transfection efficiency of SERPINH1 siRNA (Fig. 4A-4C). Next, we evaluated the effect of SERPINH1 interference on IRE1α, GRP78, XBP1-s, and XBP1-u expression in the HGECs. The expression levels of IRE1α, GRP78 and XBP1-s were significantly decreased, and those of XBP1-u and p-IRE1α were increased by SERPINH1 siRNA transfection in the HGECs, suggesting that UPR failure resulted in prolonged or severe ER stress (Fig. 4D, 4G, and 4I).
In addition, we explored the effects of SERPINH1 siRNA transfection on inflammation in HGECs. Activation of the NF-kappa B pathway plays an important role in the progression of inflammation. ER stress aggravates the inflammatory response through the IRE1α-associated NF-κB signaling pathway [27]. Therefore, we detected p-p65, NLRP3 and IL-1β expression in HGECs using RT-qPCR and western blotting. The expression levels of p-p65, NLRP3 and IL-1β were significantly increased by SERPINH1 siRNA transfection (Fig. 4D-4I). Taken together, these results indicate that silencing SERPINH1 significantly prolonged ER stress responses and initiated or aggravated an abnormal inflammatory response in HGECs.
SERPINH1 overexpression regulated the ER stress response and alleviated inflammation in HGECs cultured under high-glucose conditions
The results of in vitro SERPINH1 silencing and the decreased expression levels of SERPINH1 in the human gingival epithelium under periodontitis with diabetes mellitus prompted us to assess the therapeutic potential of SERPINH1 overexpression in an in vitro model. We hypothesized that SERPINH1 overexpression would ameliorate prolonged ER stress and the inflammatory response. To further investigate gingival epithelial barrier function and inflammatory responses, HBLV-h-SERPINH1-transfected HGECs were successfully constructed, and the mRNA and protein expression levels of SERPINH1 were significantly upregulated in the HBLV-h-SERPINH1 HGECs (Fig. 5A-5D). Next, we detected the expression levels of related genes and proteins, including SERPINH1, IRE1α, p-IRE1α, GRP78, XBP1-s, XBP1-u, p-p65, NLRP3, and IL-1β, by RT-qPCR and western blot analysis to evaluate the ability of SERPINH1 to alleviate the prolonged ER stress response and inflammation. In the high-glucose group compared to the group without high-glucose treatment, the expression levels of XBP1-u, p-IRE1α, p-p65, NLRP3, and IL-1β were higher, and the expression levels of SERPINH1, IRE1α, GRP78, XBP1-s were lower. However, following treatment with HBLV-h-SERPINH1 under high glucose, the expression levels of XBP1-u, p-IRE1α, p-p65, NLRP3, and IL-1β were significantly decreased, and the expression levels of SERPINH1, IRE1α, GRP78, and XBP1-s were significantly increased compared with those in the high-glucose group. (Fig. 5E-5K). Our results proved that SERPINH1 overexpression mitigated the prolonged ER stress response and inflammation in HGECs under high-glucose conditions.
Collectively, in vitro SERPINH1-knockdown and SERPINH1-overexpression experiments indicated the broad applicability of SERPINH1 overexpression as it diminished the inflammatory response, which may play a role in the development of periodontitis with diabetes mellitus, in HGECs under high-glucose conditions and that the IRE1α signaling pathway may be involved in regulating inflammation in HGECs under high-glucose conditions.