Risk factors for pulmonary complications after colorectal cancer surgery: a Japanese multicenter study.

PURPOSE: Pulmonary complications (PC) are a serious condition with a 20% mortality rate. However, few reports have examined risk factors for PC after colorectal surgery. This study investigated the frequency, characteristics, and risk factors for PC after colorectal cancer surgery. METHODS: Between January 2016 and December 2022, we retrospectively reviewed 3979 consecutive patients who underwent colorectal cancer surgery in seven participating hospitals. Patients were divided into patients who experienced PC (PC group, n = 54) and patients who did not (non-PC group, n = 3925). Clinical and pathological features were compared between groups. RESULTS: Fifty-four patients (1.5%) developed PC, of whom 2 patients (3.7%) died due to PC. Age was greater (80 years vs 71 years; p < 0.001), frequency of chronic obstructive pulmonary distress was greater (9.3% vs 3.2%; p = 0.029), performance status was poorer (p < 0.001), the proportion of underweight was higher (42.6% vs 13.4%, p < 0.001), frequency of open surgery was greater (24.1% vs 9.3%; p < 0.001), and blood loss was greater (40 mL vs 22 mL; p < 0.001) in the PC group. Multivariate analysis revealed male sex (odds ratio (OR) 2.165, 95% confidence interval (CI) 1.176-3.986; p = 0.013), greater age (OR 3.180, 95%CI 1.798-5.624; p < 0.001), underweight (OR 3.961, 95%CI 2.210-7.100; p < 0.001), and poorer ASA-PS (OR 3.828, 95%CI 2.144-6.834; p < 0.001) as independent predictors of PC. CONCLUSION: Our study revealed male sex, greater age, underweight, and poorer ASA-PS as factors associated with development of PC, and suggested that pre- and postoperative rehabilitation and pneumonia control measures should be implemented for patients at high risk of PC.

Short- and Long-term Outcomes After Colonic Stent Insertion as a Bridge to Surgery in Elderly Colorectal Cancer Patients.

BACKGROUND/AIM: Colonic stents have been inserted as a bridge to surgery in patients with resectable colorectal cancer, allowing bowel decompression for systemic assessment and better preparation to avoid stoma construction. However, reports of short- and long-term prognoses for elderly patients remain limited. PATIENTS AND METHODS: This retrospective study reviewed 175 consecutive patients who underwent colonic stent insertion for bowel obstruction followed by curative colectomy. Patients were divided into those >80 years old (Old, n=49) and those <80 years old (Young, n=126). After propensity score matching, 41 patients in each group matched. RESULTS: Before matching, performance status was poorer (p<0.001), postoperative complication rate was higher (p=0.009), adjuvant chemotherapy rate was lower (p<0.001), and hospital stay was longer (p<0.001) in the Old group. After matching, adjuvant chemotherapy rate was lower (9.8% vs. 39.0%; p=0.003) and hospital stay was longer (14 vs. 12 days; p=0.029) in the Old group. Five-year relapse-free survival (42.9% vs. 68.8%; p=0.200), overall survival (66.3% vs. 87.7%; p=0.081), and cancer-specific survival (68.2% vs. 87.7%; p=0.129) rates were comparable between groups. CONCLUSION: Colorectal resection after colonic stent insertion is useful for elderly patients, with potential to reduce postoperative complication rates and achieve good long-term results with appropriate case selection.

Impact of institutional volume on short- and long-term outcomes after laparoscopic colectomy.

INTRODUCTION: The impact of institutional volume on postoperative outcomes after laparoscopic colectomy is still being debated. This study aimed to investigate whether differences in postoperative outcomes of laparoscopic colon resection exist between high- and low-volume centers. METHODS: Data were reviewed for 1360 patients who underwent laparoscopic colectomy for colon cancer between 2016 and 2022. Patients were divided according to whether they were treated at a high-volume center (≥100 colorectal surgeries annually; n = 947) or a low-volume center (<100 colorectal surgeries annually; n = 413). Propensity score matching was applied to balance covariates and minimize selection biases that could affect outcomes. Finally, 406 patients from each group were matched. RESULTS: After matching, patients from high-volume centers showed a higher number of retrieved lymph nodes (19 vs. 17, p < .001) and more frequent involvement of expert surgeons (98.3% vs. 88.4%, p < .001). Postoperative complication rates were similar between groups (p = .488). No significant differences between high- and low-volume centers were seen in relapse-free survival (88.8% each, p = .716) or overall survival (85.7% vs. 82.8%, p = .480). CONCLUSION: The present study suggests that in appropriately educated organizations, relatively safe procedures and good prognosis may be obtained for laparoscopic colectomy cases, regardless of institutional volume.

A case of pancreaticoduodenal artery bleeding after laparoscopic right colectomy requiring open hemostasis.

BACKGROUND: Pancreatic and duodenal-related complications after right colectomy carry a higher risk of mortality. CASE PRESENTATION: A 64-year-old woman underwent laparoscopic right colectomy for a laterally spreading tumor in the cecum. On postoperative day 10, she experienced sudden hematemesis. Contrast-enhanced computed tomography (CT) of the abdomen showed a large amount of hemorrhage in the stomach, but no obvious extravasation. In addition, free air was observed near the duodenal bulb. Despite blood transfusion, vital signs remained unstable and emergency surgery was performed. The abdomen was opened through midline incisions in the upper and lower abdomen. A fragile wall and perforation were observed at the border of the left side of the duodenal bulb and pancreas, with active bleeding observed from inside. As visualization of the bleeding point proved difficult, the duodenum was divided circumferentially to confirm the bleeding point and hemostasis was performed using 4-0 PDS. The left posterior wall of the duodenum was missing, exposing the pancreatic head. For reconstruction, the jejunum was elevated via the posterior colonic route and the duodenal segment and elevated jejunum were anastomosed in an end-to-side manner. Subsequently, gastrojejunal and Brown anastomoses were added. Drains were placed before and after the duodenojejunal anastomosis. Postoperative vital signs were stable and the patient was extubated on postoperative day 1. Follow-up contrast-enhanced CT of the abdomen showed no active bleeding, and the patient was discharged home on postoperative day 21. As of 6 months postoperatively, the course of recovery has been uneventful. CONCLUSIONS: We encountered a case of pancreaticoduodenal artery hemorrhage after laparoscopic right colectomy. Bleeding at this site can prove fatal, so treatment plans should be formulated according to the urgency of the situation.

Intestinal Behçet's and suspected intestinal Behçet's disease: a report of four surgical cases.

BACKGROUND: Intestinal Behçet's disease (BD) is often associated with ulceration that requires surgery, including perforation and abscess formation. However, no consensus has been reached on the optimal extent of resection or treatment strategy. This study reviewed four cases of intestinal or suspected intestinal BD. CASE PRESENTATIONS: In Case 1, a 74-year-old woman diagnosed with BD 2 years earlier was treated with anti-tumor necrosis factor α antibody (Infliximab) and steroids. She had oral and pubic ulcers. After close investigation of abdominal pain, perforation of the gastrointestinal tract was suspected and surgery was performed. Multiple perforating ulcers and abscesses were found in the distal ileum, and the small intestine was resected. Postoperatively, the patient was treated with an increased steroid dose and symptoms have remained stable. Case 2 involved a 69-year-old woman with oral and pubic ulcers, ocular ulcer, and skin lesions. She experienced sudden onset of abdominal pain during treatment for lymphoma. She showed multiple perforating ulcers throughout the ileum and underwent resection of the small intestine and ileostomy. Upper abdominal pain appeared during postoperative treatment for high-output syndrome. The patient underwent omentoplasty after perforation of the upper gastrointestinal tract was diagnosed. Postoperatively, anti-interleukin-1 beta antibodies (canakinumab) was administered to control the disease. Case 3 involved an 81-year-old, previously healthy woman. She presented to her previous physician with complaints of pubic ulcer, hemorrhage and abdominal pain. Colonoscopy showed multiple ulcers throughout the entire colon. Steroid therapy was started, but bleeding proved difficult to control and total proctocolectomy was performed. Histopathology revealed multiple perforating ulcers and BD was diagnosed. Postoperatively, the patient remains under steroid control. Case 4 involved a 43-year-old man with abdominal pain who showed abscess formation in the ileocecal region. After excision of the ileocecal area, multiple ulcers were diagnosed. Two years later, abdominal pain recurred and free air was found in the abdomen on close imaging. Emergency anastomotic resection was performed due to ulceration and perforation of the anastomosis. CONCLUSIONS: Intestinal BD may flare up after surgical treatment and require multiple surgeries. Introducing pharmacotherapy as soon as possible after surgical treatment is important to control the disease.

Prognostic value of lymph node distribution after laparoscopic colectomy with Japanese D3 dissection.

PURPOSE: Positive pathologic lymph nodes in colorectal cancer represent an important prognostic indicator. Whether lymph node distribution or the number of metastatic nodes is more strongly associated with survival prediction remains controversial. METHODS: Among 3449 colorectal cancer surgeries performed at Nagasaki University Hospital and five affiliated institutions from April 2016 to March 2022, we investigated 604 patients who underwent laparoscopic radical resection and were diagnosed with pathological stage III cancer. Patients were divided into two groups according to whether they had central vessel metastasis (LND3 group, n=42) or not (LND1/2 group, n=562). After adjusting for background factors using propensity score matching, the LND3 group included 42 patients and the LND1/2 group included 40 patients. Patient background characteristics and prognosis were compared between these two groups. RESULTS: Before matching, frequencies of right-side colon cancer (64.3% vs 38.1%, p=0.001), multivisceral resection (11.9% vs 4.4%, p=0.039), clinical N2 status (40.5% vs 22.6%, p=0.032), and pathological N2 (73.8% vs 22.6%, p<0.001) were all greater, and the number of lymph nodes retrieved was higher (24 vs 19, p=0.042) in the LND3 group. After matching, no differences in any clinical factors were evident between groups. Five-year RFS (44.8% vs 77.1%, p=0.004) and OS (43.1% vs 83.2%, p<0.001) were worse in the LND3 group. Adjuvant chemotherapy improved RFS (adjuvant chemotherapy (+) vs adjuvant chemotherapy (-): 62.1% vs 27.7%, p=0.047) in the LND3 group. CONCLUSION: LND3-positive patients show poorer prognosis than LND1/2 patients and should be treated with an appropriate perioperative treatment strategy.

Correlations of age with clinicopathological features, perioperative outcomes and the prognosis in patients with colorectal cancer: a Japanese multicenter study.

PURPOSE: Colorectal cancer is not common in patients under 40 years old, and its associations with clinical features and the prognosis remain uncertain. METHODS: Using a multicenter database, we retrospectively reviewed 3015 patients who underwent colorectal surgery between 2016 and 2021. Patients were divided by age into those < 40 years old (young; n = 52), 40-54 years old (middle-aged; n = 254) and > 54 years old (old; n = 2709). We then investigated age-related differences in clinicopathological features, perioperative outcomes and the prognosis. RESULTS: The proportion of young patients increased annually from 0.63% in 2016 to 2.10% in 2021. Female patients were more frequent, the performance status was better, tumors were larger, clinically node-positive and poorly differentiated adenocarcinomas were more frequent, postoperative complications were less frequent, and the hospital stay was shorter in young patients than in older patients. Young age was an independent predictor of a low risk of postoperative complications (odds ratio, 0.204; 95% confidence interval, 0.049-0.849; p = 0.028). With pathologically node-positive status, adjuvant chemotherapy was more frequent in young patients (100%) than in middle-aged (73.7%) or old (51.8%) patients (p < 0.001), and the 3-year relapse-free survival was better in the young group than in others. CONCLUSION: Despite higher rates of advanced tumors in younger patients, adequate adjuvant chemotherapy appears to improve the relapse-free survival.

Risk factors for reoperation within 30 days in laparoscopic colorectal cancer surgery: A Japanese multicenter study.

INTRODUCTION: Thirty-day reoperation rate reflects short-term surgical outcomes following surgery. Laparoscopic surgery for colorectal cancer reportedly has positive effects on postoperative complications. This retrospective study investigated risk factors for 30-day reoperation rate among patients after laparoscopic colorectal cancer surgery using a multicenter database. METHODS: Participants comprised 3037 patients who had undergone laparoscopic resection of colorectal cancer between April 2016 and December 2022 at the Nagasaki University and six affiliated centers, classified into those who had undergone reoperation within 30 days after surgery (RO group; n = 88) and those who had not (NRO group; n = 2949). Clinicopathological characteristics were compared between groups. RESULTS: In the RO group, anastomotic leakage occurred in 57 patients (64.8%), intestinal obstruction in 12 (13.6%), and intraabdominal abscess in 5 (5.7%). Female patients were more frequent, preoperative treatment less frequent, body mass index (BMI) lower, operation time longer, blood loss greater, and hospital stay longer in the RO group (p < .05 each). Multivariate analysis revealed BMI (odds ratio, 0.415; 95% confidence interval, 0.218-0.787; p = .021) and poor performance status (odds ratio, 1.966; 95% confidence interval, 1.106-3.492; p = .021) as independent predictors of reoperation. CONCLUSION: Perioperative measures are warranted for patients with low BMI and poor performance status undergoing laparoscopic colorectal surgery.

Long-term outcomes and early recurrence after resection for metachronous pulmonary metastases from colorectal cancer.

PURPOSE: Properly selecting patients for aggressive curative resection for pulmonary metastases (PMs) from colorectal cancer (CRC) is desirable. We purposed to clarify prognostic factors and risk factors for early recurrence after metachronous PM resection. METHODS: Clinical data of 151 patients who underwent R0 resection for metachronous PMs from CRC at two institutions between 2008 and 2021 were reviewed. RESULTS: Seventy-six patients (50.3%) were male, and the median age was 71 (42-91) years. The numbers of colon/rectal cancers were 76/75, with pStage I/II/III/IV/unknown in 15/34/86/13/3. The duration from primary surgery to PM was 19.7 (1.0-106.4) months. The follow-up period was 41.9 (0.3-156.2) months. The 1-, 3-, and 5-year recurrence-free survival (RFS) rates were 75.1%, 53.7%, and 51.1%, and the 1-, 3-, and 5-year overall survival (OS) rates were 97.7%, 87.5%, and 68.2%. On multivariate analysis, lymph node metastasis of the primary lesion (HR 1.683, 95%CI 1.003-2.824, p = 0.049) was an independent predictor of poor RFS, and history of resection for extrapulmonary metastasis (e-PM) (HR 2.328, 95%CI 1.139-4.761, p = 0.021) was an independent predictor of poor OS. Patients who experienced early recurrence (< 6 months) after PM resection showed poorer OS than others (3-year OS 50.8% vs. 90.2%, p = 0.002). On multivariate analysis, e-PM was an independent predictor of early recurrence after PM resection (OR 3.989, 95%CI 1.002-15.885, p = 0.049). CONCLUSION: Since a history of e-PM was a predictor of early recurrence and poor OS after R0 resection for PM, surgical treatment of patients with a history of e-PM should be considered carefully.

Malignant peripheral nerve sheath tumor in the pelvis: a case report.

BACKGROUND: Malignant peripheral nerve sheath tumors (MPNSTs) are malignancies that arise or differentiate from or infiltrate peripheral nerves and account for approximately 5% of soft-tissue malignancies. Approximately half of MPNSTs develop in patients with neurofibromatosis type 1 (NF1), a hereditary disease. MPNSTs occur mainly in the trunk, proximal extremities, and neck, but can on rare occasion arise in or near the gastrointestinal tract, and intestinal complications have been reported. We describe herein a case with resection of an MPNST arising in the pelvic region. CASE PRESENTATION: A 51-year-old woman had undergone repeated resections for systemic neurofibrosis associated with NF1. This time, a pelvic tumor was noted on follow-up positron emission tomography computed tomography (CT). She presented with slowly progressive radiating pain in the lower extremities and was referred to our hospital for tumor resection. Contrast-enhanced CT showed a 75 × 58-mm mass in the right greater sciatic foramen directly below a 24 × 28-mm mass. Open pelvic tumor resection was performed for pelvic neurofibroma. The obturator nerve was identified lateral to the main tumor and the sciatic nerve was identified dorsally, then dissection was performed. The closed nerve was spared, while the sciatic nerve was partially dissected and the two tumors were removed. Both tumors were elastic and hard. Pathologic findings were MPNST for the large specimen and neurofibroma with atypia for the small specimen. The patient developed temporary postoperative ileus, but is generally doing well and is currently free of recurrence or radiating pain. The patient is at high risk of recurrence and close monitoring should be continued. CONCLUSIONS: We encountered a rare case of MPNST. Due to the high risk of recurrence, surgery with adequate margins was performed, with a requirement for appropriate follow-up.

Effect of adjuvant chemotherapy after curative resection of colorectal cancer peritoneal metastasis.

PURPOSE: Several guidelines have recommended surgical resection for localized peritoneal metastases, but the prognosis remains poor. In addition, the efficacy of adjuvant chemotherapy (AC) after curative resection is under debate. The present study compared long-term outcomes between curative and non-curative resection and evaluated the effects of AC after curative resection. METHODS: Using a multicenter database, we retrospectively reviewed 123 colorectal cancer patients with peritoneal metastases between April 2016 and December 2021. Of these patients, 49 underwent curative resection, and 74 underwent non-curative resection. RESULTS: The frequency of broad metastases was lower in the curative resection group (8.2%) than in the non-curative resection group (43.2%, p < 0.001). Among all patients, 5-year overall survival rate was higher in the curative resection group (43.0%) than in the non-curative resection group (7.3%, p = 0.004). Among patients who underwent curative resection, 5-year overall survival rate was significantly higher in the AC group (48.2%) than in the non-AC group (38.1%, p = 0.037). Multivariate analysis of all patients revealed pathological N status and non-curative resection as independent predictors of overall survival. In patients who underwent curative resection, advanced age was an independent predictor of relapse-free survival, and AC was an independent predictor of overall survival. CONCLUSION: This multicenter study of colorectal cancer patients with peritoneal metastases revealed that prognosis was more favorable for curable cases than for non-curable cases. Prognosis was more favorable in the AC group than in the non-AC group after curative resection.

Aberrant common hepatic artery from the left gastric artery and with no connection to the gastroduodenal artery: Risky vascular anomaly in gastrectomy.

Knowledge of anatomical variations of the celiac axis is important in upper abdominal surgery. Aberrant common hepatic artery originating from the left gastric artery without connecting the gastroduodenal artery is extremely rare. Preoperative vascular anatomy assessment using reconstructions of CT images may be useful for safe surgical procedure.

MiR-200b suppresses TNF-α-induced AMTN production in human gingival epithelial cells.

Amelotin (AMTN) is an enamel protein that is localized in junctional epithelium (JE) of gingiva and suggested to be involved in the attachment between JE and tooth enamel. MicroRNA is a small non-coding RNA that regulates gene expression at post-transcriptional level by binding to the 3'-untranslated region (3'-UTR) of target mRNAs. In this study, we have analyzed the effects of miR-200b on the expression of AMTN in human gingival epithelial (Ca9-22) cells. Total RNAs and proteins were extracted from Ca9-22 cells transfected with miR-200b expression plasmid or miR-200b inhibitor and stimulated by TNF-α (10 ng/ml, 12 h). AMTN and inhibitor of kappa-B kinase beta (IKKß) mRNA and protein levels were measured by qPCR and Western blot. Human AMTN 3'-UTR that contains putative miR-200b target sites were cloned downstream of -353AMTN luciferase (LUC) plasmid. Ca9-22 cells were transfected with -353AMTN 3'-UTR LUC constructs and miR-200b expression plasmid, and LUC activities were measured with or without stimulation by TNF-α. TNF-α-induced AMTN mRNA levels were partially inhibited by miR-200b overexpression and enhanced by miR-200b inhibitor. TNF-α-induced IKKß mRNA and protein levels were almost completely inhibited by miR-200b. Transcriptional activities of -353AMTN 3'-UTR LUC constructs were induced by TNF-α and partially inhibited by miR-200b. IKKß inhibitor IMD0354 and NF-κB inhibitor triptolide decreased TNF-α-induced LUC activities. Furthermore, both inhibitors reduced AMTN mRNA levels in the presence or absence of TNF-α. These results suggest that miR-200b suppresses AMTN expression by targeting to AMTN and IKKß mRNAs in the human gingival epithelial cells.

Nondegenerated cystic neuroendocrine tumor of the pancreas: a case report.

BACKGROUND: Pancreatic neuroendocrine tumors (PNETs) are typically solid neoplasms but, in very rare cases, present as cystic lesions. We describe a case of a cystic neuroendocrine tumor that developed as a small cystic lesion. CASE PRESENTATION: In 2011, a 66-year-old Japanese woman underwent computed tomography (CT) that revealed a cystic lesion in the tail of the pancreas measuring 9 mm. She did not have any symptoms. She underwent a CT scan every year thereafter. The cystic lesion gradually increased and was 40 mm in 2019; endoscopic retrograde pancreatography (ERP) was then performed. Cytological examination demonstrated class IIIb adenocarcinoma, and we conducted laparoscopic distal pancreatectomy. Pathological examination showed PNET. CONCLUSION: Although cystic change of PNET is generally caused by ischemia or necrosis inside the tumor, in our case, PNET occurred as a small cyst that increased without changing form.

Factors related to short-term outcomes and delayed systemic treatment following primary tumor resection for asymptomatic stage IV colorectal cancer.

BACKGROUND: The effectiveness of primary tumor resection (PTR) for asymptomatic stage IV colorectal cancer patients to continue prolonged and safe systemic chemotherapy has recently been re-evaluated. However, postoperative complications lead to a prolonged hospital stay and delay systemic treatment, which could result in a poor oncologic outcome. The objective of this study was to identify the risk factors for morbidity and delay of systemic chemotherapy in such patients. METHODS: Between April 2016 and March 2018, 115 consecutive colorectal cancer patients with distant metastasis who had no clinical symptoms and underwent PTR in all participating hospitals were retrospectively reviewed. The patients were divided into two groups according to the presence (CD ≥ 2, n = 23) or absence (CD < 2, n = 92) of postoperative complications. RESULTS: The proportion of combined resection of adjacent organs was significantly higher in the postoperative complication group (p = 0.014). Complications were significantly correlated with longer hospital stay (p < 0.001) and delay of first postoperative treatment (p = 0.005). Univariate and multivariate analyses showed that combined resection (odds ratio 4.593, p = 0.010) was the independent predictor for postoperative complications. Median survival time was 8.5 months. Postoperative complications were not associated with overall survival, but four patients (3.5%) could not receive systemic chemotherapy because of prolonged postoperative complications. CONCLUSIONS: Although PTR for asymptomatic stage IV CRC patients showed an acceptable prognosis, appropriate patient selection is needed to obtain its true benefit.

Follicular dendritic cell-secreted protein gene expression is upregulated and spread in nifedipine-induced gingival overgrowth.

Follicular dendritic cell-secreted protein (FDC-SP) is secreted protein expressed in follicular dendritic cells, periodontal ligament and junctional epithelium (JE). Its expression could be controlled during inflammatory process of gingiva; however, responsible mechanism for gingival overgrowth and involvement of FDC-SP in clinical condition is still unclear. We hypothesized that JE-specific genes are associated with the initiation of drug-induced gingival enlargement (DIGE) called gingival overgrowth, and investigated the changes of JE-specific gene's expression and their localization in overgrown gingiva from the patients. Immunohistochemical analysis revealed that the FDC-SP localization was spread in overgrown gingival tissues. FDC-SP mRNA levels in GE1 and Ca9-22 cells were increased by time-dependent nifedipine treatments, similar to other JE-specific genes, such as Amelotin (Amtn) and Lamininß3 subunit (Lamß3), whereas type 4 collagen (Col4) mRNA levels were decreased. Immunocytochemical analysis showed that FDC-SP, AMTN, and Lamß3 protein levels were increased in GE1 and Ca9-22 cells. Transient transfection analyses were performed using luciferase constructs including various lengths of human FDC-SP gene promoter, nifedipine increased luciferase activities of -345 and -948FDC-SP constructs. These results raise the possibility that the nifedipine-induced FDC-SP may be related to the mechanism responsible for gingival overgrowth does not occur at edentulous jaw ridges.

C/EBPß and YY1 bind and interact with Smad3 to modulate lipopolysaccharide-induced amelotin gene transcription in mouse gingival epithelial cells.

Junctional epithelium (JE) develops from reduced enamel epithelium during tooth formation and is critical for the maintenance of healthy periodontal tissue through ensuring appropriate immune responses and the rapid turnover of gingival epithelial cells. We have previously shown a relationship between inflammatory cytokines and expression of JE-specific genes, such as amelotin (AMTN), in gingival epithelial cells. Here, we elucidated the effects of Porphyromonas gingivalis-derived lipopolysaccharide (Pg LPS) on Amtn gene transcription and the interaction of transcription factors. To determine the molecular basis of transcriptional regulation of the Amtn gene by Pg LPS, we performed real-time PCR and carried out luciferase assays using a mouse Amtn gene promoter linked to a luciferase reporter gene in mouse gingival epithelial GE1 cells. Gel mobility shift and chromatin immunoprecipitation assays were performed to identify response elements bound to LPS-induced transcription factors. Next, we analyzed protein levels of the LPS-induced transcription factors and the interaction of transcription factors by western blotting and immunoprecipitation. LPS increased Amtn mRNA levels and elevated luciferase activities of constructs containing regions between -116 and -238 of the mouse Amtn gene promoter. CCAAT/enhancer-binding protein (C/EBP) 1-, C/EBP2- and Ying Yang 1 (YY1)-nuclear protein complexes were increased by LPS treatment. Furthermore, we identified LPS-modulated interactions with C/EBPß, YY1 and Smad3. These results demonstrate that Pg LPS regulates Amtn gene transcription via binding of C/EBPß-Smad3 and YY1-Smad3 complexes to C/EBP1, C/EBP2 and YY1 response elements in the mouse Amtn gene promoter.

Negative feedback by SNAI2 regulates TGFß1-induced amelotin gene transcription in epithelial-mesenchymal transition.

Junctional epithelium (JE) demonstrates biological responses with the rapid turnover of gingival epithelial cells. The state occurs in inflammation of gingiva and wound healing after periodontal therapy. To understand the underlying mechanisms and to maintain homeostasis of JE, it is important to investigate roles of JE-specific genes. Amelotin (AMTN) is localized at JE and regulated by inflammatory cytokines and apoptotic factors that represent a critical role of AMTN in stabilizing the dentogingival attachment, which is an entrance of oral bacteria. In this study, we demonstrated that the AMTN gene expression was regulated by SNAI2 and transforming growth factor ß1 (TGFß1)-induced epithelial-mesenchymal transition (EMT) that occurs in wound healing and fibrosis during chronic inflammation. SNAI2 downregulated AMTN gene expression via SNAI2 bindings to E-boxes (E2 and E4) in the mouse AMTN gene promoter in EMT of gingival epithelial cells. Meanwhile, TGFß1-induced AMTN gene expression was attenuated by SNAI2 and TGFß1-induced SNAI2, without inhibition of the TGFß1-Smad3 signaling pathway. Moreover, SNAI2 small interfering RNA (siRNA) rescued SNAI2-induced downregulation of AMTN gene expression, and TGFß1-induced AMTN gene expression was potentiated by SNAI2 siRNA. Taken together, these data demonstrated that AMTN gene expression in the promotion of EMT was downregulated by SNAI2. The inhibitory effect of AMTN gene expression was an independent feedback on the TGFß1-Smad3 signaling pathway, suggesting that the mechanism can be engaged in maintaining homeostasis of gingival epithelial cells at JE and the wound healing phase.

Effects of interleukin-1ß on human follicular dendritic cell-secreted protein gene expression in periodontal ligament cells.

Follicular dendritic cell-secreted protein (FDC-SP) is expressed in FDCs, human periodontal ligament (HPL) cells, and junctional epithelium. To evaluate the effects of interleukin-1 beta (IL-1ß) on FDC-SP gene expression in immortalized HPL cells, FDC-SP mRNA and protein levels in HPL cells following stimulation by IL-1ß were measured by real-time polymerase chain reaction and Western blotting. Luciferase (LUC), gel mobility shift, and chromatin immunoprecipitation (ChIP) analyses were performed to study the interaction between transcription factors and promoter regions in the human FDC-SP gene. IL-1ß (1 ng/mL) induced the expression of FDC-SP mRNA and protein levels at 3 h, and reached maximum levels at 12 h. IL-1ß increased LUC activities of constructs (-116FDCSP - -948FDCSP) including the FDC-SP gene promoter. Transcriptional inductions by IL-1ß were partially inhibited by 3-base-pair (3-bp) mutations in the Yin Yang 1 (YY1), GATA, CCAAT-enhancer-binding protein2 (C/EBP2), or C/EBP3 in the -345FDCSP. IL-1ß-induced -345FDCSP activities were inhibited by protein kinase A, tyrosine-kinase, mitogen-activated protein kinase (MEK)1/2, and PI3-kinase inhibitors. The results of gel shift and ChIP assays revealed that YY1, GATA, and C/EBP-ß interacted with the YY1, GATA, C/EBP2, and C/EBP3 elements that were increased by IL-1ß. These studies demonstrate that IL-1ß increases FDC-SP gene transcription in HPL cells by targeting YY1, GATA, C/EBP2, and C/EBP3 in the human FDC-SP gene promoter.

IL-1ß and TNF-α regulate mouse amelotin gene transcription in gingival epithelial cells.

Amelotin (AMTN) is an enamel protein expressed in maturation-stage ameloblasts and junctional epithelium. To clarify the transcriptional regulation of the AMTN gene by interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α), we conducted real-time PCR, Western blotting, transient transfection analyses with luciferase constructs including various lengths of the mouse AMTN gene promoter, and gel shift and chromatin immunoprecipitation assays using mouse gingival epithelial GE1 cells. The levels of AMTN mRNA and protein in GE1 cells were increased after 6 h of stimulation with IL-1ß (1 ng/mL) and TNF-α (10 ng/mL). IL-1ß and TNF-α induced luciferase activities of the constructs between -116AMTN and -705AMTN including the mouse AMTN gene promoter. Transcriptional activation by IL-1ß and TNF-α was partially inhibited in -460AMTN including 3-bp mutations in the CCAAT-enhancer-binding protein 1 (C/EBP1), C/EBP2 and Yin Yang 1 (YY1) elements. Transcriptional activities induced by IL-1ß and TNF-α were inhibited by tyrosine kinase, MEK1/2 and PI3-kinase inhibitors. Results of ChIP assays showed that IL-1ß and TNF-α increased C/EBPß and YY1 binding to the C/EBP1, C/EBP2 and YY1 elements. These results demonstrate that IL-1ß and TNF-α increase AMTN gene transcription via the C/EBP1, C/EBP2 and YY1 elements in the mouse AMTN gene promoter.