Early induction of Hes1 by bone morphogenetic protein 9 plays a regulatory role in osteoblastic differentiation of a mesenchymal stem cell line.

Bone morphogenic protein 9 (BMP9) is one of the most potent inducers of osteogenic differentiation among the 14 BMP members, but its mechanism of action has not been fully demonstrated. Hes1 is a transcriptional regulator with basic helix-loop-helix (bHLH) domain and is a well-known Notch effector. In this study, we investigated the functional roles of early induction of Hes1 by BMP9 in a mouse mesenchymal stem cell line, ST2. Hes1 mRNA was transiently and periodically induced by BMP9 in ST2, which was inhibited by BMP signal inhibitors but not by Notch inhibitor. Interestingly, Hes1 knockdown in ST2 by siRNA increased the expression of osteogenic differentiation markers such as Sp7 and Ibsp and matrix mineralization in comparison with control siRNA transfected ST2. In contrast, forced expression of Hes1 by using the Tet-On system suppressed the expression of osteogenic markers and matrix mineralization by BMP9. We also found that the early induction of Hes1 by BMP9 suppressed the expression of Alk1, an essential receptor for BMP9. In conclusion, BMP9 rapidly induces the expression of Hes1 via the SMAD pathway in ST2 cells, which plays a negative regulatory role in osteogenic differentiation of mesenchymal stem cells induced by BMP9.

Lipoma in Parotid Gland: A Rare Case Report.

Lipoma is a benign, rare, mesenchymal tumor found in the head and neck region, especially in the parotid gland. It thus requires a careful and precise examination to establish a diagnosis. A surgical procedure of the parotid gland is challenging due to the associated risk factor of facial nerve injury. We report a rare case of head and neck region lipoma between the superficial and deep lobe of the parotid gland. A 44-year-old female patient was presented with the chief complaint of a painless lump on the left front ear to the left cheek for about 1 year. There were no complaints of tooth pain before the lump appeared, and there were no lumps in other regions. A fine-needle aspiration biopsy, ultrasonography, and magnetic resonance imaging were all performed to establish the preoperative diagnosis and to plan the correct surgical approach. Lipoma was the initial clinical diagnosis, and a surgical excision with superficial parotidectomy and facialis nerve preservation was performed. Follow-up examinations were conducted to assess any facial nerve injury complications. Conclusion Lipoma rarely grows in the parotid gland. Careful diagnosis should be performed to establish a precise surgery for parotid dissection and facial nerve preservation.

The contradictory effects of coffee intake on periodontal health: a systematic review of experimental and observational studies.

Background: Drinking coffee is known to have both positive and negative aftermath on periodontal health. The current study is aiming to systematically review the impact of coffee consumption on periodontal health status. Methods: An article search was carried out in two electronic databases (PUBMED and Web of Sciences). All type of experimental and observational studies were included. The assessment of the included articles were conducted using Joanna Briggs Institute (JBI) critical appraisal tool. Data were analyzed qualitatively. Result: A total of 10 articles were included in this study. Most (5) of the studies discovered a negative correlation between coffee intake and periodontal health, while 4 other studies found the protective effect of daily coffee consumption against alveolar bone loss. Last, only one study found that coffee intake did not relate with periodontitis. Conclusion: The effect of coffee consumption on periodontal health was fragmented since coffee has complex components that may give either beneficial effects or negative impact on periodontal health.

Three-Dimensional Quantification of Postoperative Facial Asymmetry in Patients With Unilateral Cleft lip and Palate Using Facial Symmetry Plane.

OBJECTIVE: To quantitatively assess three-dimensional (3D) soft tissue facial asymmetry in patients with unilateral cleft lip and palate (UCLP) who have undergone primary lip repair. DESIGN: Clinical, retrospective, comparative, methodological study. PATIENTS/PARTICIPANTS: Twenty patients with UCLP were selected after a review of the records. INCLUSION CRITERIA: Complete UCLP; surgically treated without secondary repair. An age-matched and sex-matched Control group was employed. MAIN OUTCOME MEASURES: A 3D facial symmetry plane (FSP) was obtained by superimposing the point clouds of the original 3D facial image excluding the surgical site and including lip and nose areas and those of a mirrored facial image using the iterative closest point (ICP) adjustment method. The discrepancies in the depth and angle of the normal vector of the facial surface of each point cloud between right and left sides (cleft and non-cleft sides in the UCLP group, respectively) based on FSP were calculated. RESULTS: Facial asymmetry in the UCLP group was significantly greater than in the Control group regarding both the discrepancies in the depth (1.34 ± 0.62, 0.73 ± 0.32 pixels, respectively) (P = .0004) and surface angle (18.0 ± 5.88, 12.8 ± 4.0°, respectively) (P = .0024). Biaxial assessment of the discrepancies in the depth and surface angle allowed us to visually extract UCLP patients with greater facial asymmetry. CONCLUSIONS: Facial asymmetry analysis based on 3D FSP effectively facilitates the facial asymmetry quantification and soft tissue surgical outcome evaluation in patients with UCLP.

HIF-1α plays an essential role in BMP9-mediated osteoblast differentiation through the induction of a glycolytic enzyme, PDK1.

Bone homeostasis is regulated by bone morphogenic proteins (BMPs), among which BMP9 is one of the most osteogenic. Here, we have found that BMP9 rapidly increases the protein expression of hypoxia-inducible factor-1α (HIF-1α) in osteoblasts under normoxic conditions more efficiently than BMP2 or BMP4. A combination of BMP9 and hypoxia further increased HIF-1α protein expression. HIF-1α protein induction by BMP9 is not accompanied by messenger RNA (mRNA) increase and is inhibited by the activation of prolyl hydroxylase domain (PHD)-containing protein, indicating that BMP9 induces HIF-1α protein expression by inhibiting PHD-mediated protein degradation. BMP9-induced HIF-1α protein increase was abrogated by inhibitors of phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT) kinase, indicating that it is mediated by PI3K-AKT signaling pathway. BMP9 increased mRNA expression of pyruvate dehydrogenase kinase 1 (PDK1), a glycolytic enzyme, and vascular endothelial growth factor-A (VEGF-A), an angiogenic factor, in osteoblasts. Notably, BMP9-induced mRNA expression of PDK1, but not that of VEGF-A, was significantly inhibited by small interference RNA-mediated knockdown of Hif-1α. BMP9-induced matrix mineralization and osteogenic marker gene expressions were significantly inhibited by chemical inhibition and gene knockdown of either Hif-1α or Pdk-1, respectively. Since increased glycolysis is an essential feature of differentiated osteoblasts, our findings indicate that HIF-1α expression is important in BMP9-mediated osteoblast differentiation through the induction of PDK1.

Postoperative Complications of Impacted Mandibular Third Molar Extraction Related to Patient's Age and Surgical Difficulty Level: A Cross-Sectional Retrospective Study.

Mandibular third molar surgical extraction, either partially erupted or fully impacted, is the most common surgical procedure in oral and maxillofacial surgery (OMFS). However, this procedure can be associated with many postoperative complications including persistent pain, swelling, trismus, and paresthesia due to nerve injury. This study aimed to identify the correlation of postoperative complications with patient's age, sex, and surgical difficulty level. This study was a cross-sectional retrospective and single-center research conducted on patients with a history of mandibular third molar surgical extraction in the period between 2017 and 2019 at Dental and Oral Hospital Universitas Airlangga, Surabaya, Indonesia. The researchers assessed the factors of age, sex, and surgical difficulty level regarding postoperative complications on the first day of the surgery and after one week on the 7th day of it. Among 916 respondents, the majority of the sample was females (59%) and the dominant age group (60.9%) was the age group of 21-30 years while the dominant surgical difficulty level was shown by the advanced cases group (77%). The statistical analysis showed that there was a significant correlation between surgical difficulty level and postoperative complications including pain, trismus, and paresthesia on the first-day assessment. On the other hand, age was significantly related to complications like pain, swelling, and trismus on the first-week assessment. Age and surgical difficulty level were the most common risk factors of the mandibular third molar extraction postoperative complications. Dentists should take into consideration that older patients (≥51 years) and patients with complex surgical level are more vulnerable to severe postoperative complications.

Decision-Making Criteria of Odontectomy or Surgical Exposure in Impacted Maxillary Canine Based on Treatment Difficulty Index Modification.

OBJECTIVE: Canine impaction is a difficult condition to treat, and it usually necessitates a combination of surgical exposure and orthodontic traction or surgical extraction. An accurate assessment of the maxillary canine's position can help determine the severity of the impaction, the difficulty of therapy, and the treatment's prognosis. MATERIALS AND METHODS: A total of 55 impacted canines were studied and selected retrospectively. Difficulty indexes were used to measure the severity of impaction with pretreatment panoramic radiographs. STATISTICAL ANALYSIS: Pearson correlation was used to test the validity of the difficulty index modification score. Regression statistical analysis was used to evaluate any correlation between total scoring from each index with surgical treatment. RESULTS: The validity test on the variable modification index score showed a valid value (p = 0.000). According to both treatment difficulty and modification index, odontectomy group showed higher mean of total scoring than surgical exposure group. Treatment difficulty and modification index showed a significant correlation with surgical treatment (p = 0.003 and p = 0.001). CONCLUSIONS: The higher the severity of canine impaction, the greater is the possibility of odontectomy than surgical exposure. Both indexes can consider to be used in determining surgical treatment planning.

Comparison of recurrence rate and speech outcome between two different techniques for cleft palatal fistula closure: A retrospective cohort study.

The aim of this retrospective cohort study was to compare the recurrence rate and speech outcomes between two techniques for palatal fistula closure of cleft palate (CP). Patients with CP who underwent secondary palatal fistula closure using the single hinge-flap method with double-breasted mattress suture (hinge-flap group) and those who were treated with the conventional sliding palatal flap method (sliding-flap group) were retrospectively evaluated for demographic and perioperative variables. Recurrence rate of palatal fistula, perceptual speech outcomes, and nasalance scores were further reviewed in patients who met the inclusion criteria. A total of 31 patients, 21 in the hinge-flap group and 10 in the sliding-flap group, were included in this study. The fistula recurrence rate in the hinge-flap group (0%) was significantly lower than that in the sliding-flap group (30.0%) (P = 0.027). In the speech assessment, hypernasality and nasalance scores decreased post-operatively in both groups and significance was observed in the hinge-flap group (P = 0.013, P < 0.001, respectively). Articulation disorders were significantly improved in the hinge-flap group (P = 0.001). Within the limitations of the study it seems that the single hinge-flap method with double-breasted mattress suture should be preferred whenever appropriate.

In Vivo Analyses of Osteogenic Activity and Bone Regeneration Capacity of Demineralized Freeze-Dried Bovine Bone Xenograft: A Potential Candidate for Alveolar Bone Fillers.

BACKGROUND: Deproteinized bovine bone mineral (DBBM) particle is the commonly used bone graft substitute in implant surgery which is mainly osteoconductive and has very slow degradation. Demineralized freeze-dried bovine bone xenograft (DFDBBX) particle is being developed as a novel xenogeneic bone filler. OBJECTIVES: The study aimed to analyze osteogenic activity and bone-forming capacity of DFDBBX particles compared to DBBM particles in alveolar bone defects in rabbit mandibles models. Material and Methods. This study investigated bone defects whether filled with DBBM particles or DFDBBX particles or left unfilled in 30 rabbit mandibles. Specimens were processed for histology, immunohistochemistry, and micro-CT scanning. Statistical difference was set at a p value < 0.05. RESULTS: The quantitative assessment showed a significantly lower number of osteoclasts and a higher number of osteoblasts in the DFDBBX group compared to the DBBM group in 2 and 4 weeks (p < 0.05). Immunostaining analyses showed significantly higher expression of RUNX2, collagen type I, alkaline phosphatase, and osteocalcin in the DFDBBX group compared to the DBBM group in 2 and 4 weeks. Bone healing score in the DFDBBX group was comparable to the DBBM group. Micro-CT presented no significant difference in the volume percentage of the mineralized tissue in the DBBM and DFDBBX groups in spite of the different healing patterns in both groups. CONCLUSION: DFDBBX particles induced higher osteoblastic activities than DBBM particles at the early stage of healing. Meanwhile, the capacity of bone formation in DFDBBX particles was comparable with DBBM particles at the later stage of healing. Considering the limitation of this study, the results presented DFDBBX particles as potential bone filler candidates.

Ultraviolet B irradiation decreases CXCL10 expression in keratinocytes through endoplasmic reticulum stress.

Ultraviolet radiation is one of the standard treatment selections for psoriasis. interferon (IFN)-γ and IFN-γ-induced CXCL10, which are highly expressed by keratinocytes in psoriasis lesion, are therapeutic targets for psoriasis. In this study, we found that ultraviolet B (UVB) irradiation inhibited IFN-γ signaling events, including STAT1 phosphorylation and induction of CXCL10 messenger RNA (mRNA) expression in keratinocytes. IFN-γ-induced expression of CXCL10 mRNA in HaCaT cells, a human keratinocyte cell line, and human epithelial keratinocytes were also inhibited by H2 O2 or endoplasmic reticulum (ER) stress inducers. Conversely, a mixture of antioxidants, Trolox and ascorbic acid, and the ER stress inhibitor salubrinal partially counteracted the inhibitory effect of UVB on IFN-γ-induced CXCL10 mRNA expression in HaCaT cells. We also found that UVB and ER stress reduced IFN-γ receptor 1 protein levels in the plasma membrane fraction of keratinocytes. These observations suggested that ER stress and the generation of reactive oxygen species are essential for the inhibitory effect of UVB on IFN-γ-induced CXCL10 mRNA in keratinocytes.

CXCL13 is a differentiation- and hypoxia-induced adipocytokine that exacerbates the inflammatory phenotype of adipocytes through PHLPP1 induction.

Hypoxia in adipose tissue is regarded as a trigger that induces dysregulation of the secretory profile in adipocytes. Similarly, local dysregulation of adipocytokine secretion is an initial event in the deleterious effects of obesity on metabolism. We previously reported that CXCL13 is highly produced during adipogenesis, however little is known about the roles of CXCL13 in adipocytes. Here, we found that hypoxia, as modeled by 1% O2 or exposure to the hypoxia-mimetic reagent desferrioxamine (DFO) has strong inductive effects on the expression of CXCL13 and CXCR5, a CXCL13 receptor, in both undifferentiated and differentiated adipocytes and in organ-cultured white adipose tissue (WAT). CXCL13 was also highly expressed in WAT from high fat diet-fed mice. Hypoxic profile, typified by increased expression of interleukin-6 (IL-6) and plasminogen activator inhibitor-1 (PAI-1) and decreased expression of adiponectin, was significantly induced by CXCL13 treatment during adipogenic differentiation. Conversely, the treatment of adipocytes with a neutralizing-antibody against CXCL13 as well as CXCR5 knockdown by specific siRNA effectively inhibited DFO-induced inflammation. The phosphorylation of Akt2, a protective factor of adipose inflammation, was significantly inhibited by CXCL13 treatment during adipogenic differentiation. Mechanistically, CXCL13 induces the expression of PHLPP1, an Akt2 phosphatase, through focal adhesion kinase (FAK) signaling; and correspondingly we show that CXCL13 and DFO-induced IL-6 and PAI-1 expression was blocked by Phlpp1 knockdown. Furthermore, we revealed the functional binding sites of PPARγ2 and HIF1-α within the Cxcl13 promoter. Taken together, these results indicate that CXCL13 is an adipocytokine that facilitates hypoxia-induced inflammation in adipocytes through FAK-mediated induction of PHLPP1 in autocrine and/or paracrine manner.

BMP9 prevents induction of osteopontin in JNK-inactivated osteoblasts via Hey1-Id4 interaction.

Osteopontin (OPN) is an osteoblast-derived secretory protein that plays a role in bone remodeling, osteoblast responsiveness, and inflammation. We recently found that osteoblast differentiation is type-specific, with conditions of JNK inactivation inducing osteoblasts that preferentially express OPN (OPN-type). Since OPN-type osteoblasts highly express osteogenesis-inhibiting proteins and Rankl, an important inducer of osteoclastogenesis, an increased appearance of OPN-type osteoblasts may be associated with inefficient and poor-quality bone regeneration. However, whether specific osteogenic inducers can modulate OPN-type osteoblast differentiation is completely unknown. Here, we demonstrate that bone morphogenic protein 9 (BMP9) prevents induction of OPN-type osteoblast differentiation under conditions of JNK inhibition. Although JNK inactivation suppressed both BMP2- and BMP9-induced matrix mineralization and osteocalcin expression, the expression of Rankl and specific cytokines such as Gpha2, Esm1, and Sfrp1 under similar conditions was increased in all cells except those treated with BMP9. Increased expression of Id4, a critical transcriptional regulator of OPN-type osteoblast differentiation, was similarly prevented only in BMP9-treated cells. We also found that BMP9 specifically induces the expression of Hey1, a bHLH transcriptional repressor, and that Id4 inhibits the suppressive effects of Hey1 on Opn promoter activity by forming Id4-Hey1 complexes in osteoblasts. Using site-direct mutagenesis, ChIP, and immunoprecipitation, we elucidated that BMP9-induced overexpression of Hey1 can overcome the effects of Id4 and suppress OPN expression. We further found that p38 activation and JNK inactivation are involved in BMP9-induced Hey1 expression. Collectively, these data suggest that BMP9 is a unique osteogenic inducer that regulates OPN-type osteoblast differentiation.

BMP9 directly induces rapid GSK3-ß phosphorylation in a Wnt-independent manner through class I PI3K-Akt axis in osteoblasts.

Bone morphogenetic protein (BMP)9 has been reported to be the most potent BMP to induce bone formation. However, the details of BMP9-transduced intracellular signaling remain ambiguous. Here, we have investigated signal transduction mechanisms of BMP9 in comparison to BMP2, another potent inducer of bone formation, in osteoblasts. In a mouse osteoblast cell line, BMP9 induced higher mRNA levels of alkaline phosphatase (ALP) and runt-related transcription factor 2 (Runx2) than BMP2 within 2 h. Unlike BMP2, BMP9 induced rapid phosphorylation of glycogen synthase kinase 3-ß (GSK3-ß) and protein kinase B (Akt) and increased the cellular protein content of ß-catenin. BMP9 moderately increased mRNA levels of several canonical Wingless-related integration site to lower degrees than BMP2. Furthermore, BMP9-induced GSK3-ß phosphorylation was not inhibited by pretreatment with actinomycin D, cycloheximide, or Brefeldin A, indicating it is independent of Wnt protein secretion. BMP9-induced GSK3-ß phosphorylation was abrogated by Akt or class I PI3K-specific inhibitors. Moreover, inactivation of GSK3-ß by LiCl did not further promote ALP and Runx2 mRNA induction by BMP9 as significantly as that by BMP2. Notably, BMP9-induced GSK3-ß phosphorylation was inhibited by small interfering RNA against endoglin and GIPC PDZ domain-containing family, member 1. Taken together, our present findings have indicated that BMP9 directly activates GSK3ß-ß-catenin signaling pathway through class I PI3K-Akt Axis in osteoblasts, which may be essential for the potent osteoinductive activity of BMP9.-Eiraku, N., Chiba, N., Nakamura, T., Amir, M. S., Seong, C.-H., Ohnishi, T., Kusuyama, J., Noguchi, K., Matsuguchi, T. BMP9 directly induces rapid GSK3-ß phosphorylation in a Wnt-independent manner through class I PI3K-Akt axis in osteoblasts.

JNK inactivation suppresses osteogenic differentiation, but robustly induces osteopontin expression in osteoblasts through the induction of inhibitor of DNA binding 4 (Id4).

Osteoblasts are versatile cells involved in multiple whole-body processes, including bone formation and immune response. Secretory amounts and patterns of osteoblast-derived proteins such as osteopontin (OPN) and osteocalcin (OCN) modulate osteoblast function. However, the regulatory mechanism of OPN and OCN expression remains unknown. Here, we demonstrate that p54/p46 c-jun N-terminal kinase (JNK) inhibition suppresses matrix mineralization and OCN expression but increases OPN expression in MC3T3-E1 cells and primary osteoblasts treated with differentiation inducers, including ascorbic acid, bone morphogenic protein-2, or fibroblast growth factor 2. Preinhibition of JNK before the onset of differentiation increased the number of osteoblasts that highly express OPN but not OCN (OPN-OBs), indicating that JNK affects OPN secretory phenotype at the early stage of osteogenic differentiation. Additionally, we identified JNK2 isoform as being critically involved in OPN-OB differentiation. Microarray analysis revealed that OPN-OBs express characteristic transcription factors, cell surface markers, and cytokines, including glycoprotein hormone α2 and endothelial cell-specific molecule 1. Moreover, we found that inhibitor of DNA binding 4 is an important regulator of OPN-OB differentiation and that dual-specificity phosphatase 16, a JNK-specific phosphatase, functions as an endogenous regulator of OPN-OB induction. OPN-OB phenotype was also observed following LPS from Porphyromonas gingivalis stimulation during osteogenic differentiation. Collectively, these results suggest that the JNK-Id4 signaling axis is crucial in the control of OPN and OCN expression during osteoblastic differentiation.-Kusuyama, J., Amir, M. S., Albertson, B. G., Bandow, K., Ohnishi, T., Nakamura, T., Noguchi, K., Shima, K., Semba, I., Matsuguchi, T. JNK inactivation suppresses osteogenic differentiation, but robustly induces osteopontin expression in osteoblasts through the induction of inhibitor of DNA binding 4 (Id4).

Reliability of Americleft Yardstick Nasolabial Appearance Assessment With/Without Basal View for Japanese Children With Unilateral Complete Cleft Lip and Palate.

OBJECTIVE: There is debate on the reliability of the Americleft Yardstick (AY) global nasolabial appearance assessment method. The objective was to analyze the effect of the additional basal view (BV) feature on the reliability of the AY method for Japanese children with complete cleft lip and palate (CUCLP). DESIGN: Blind retrospective analysis of clinical records on 43 patients (5- to 7-year-old) with nonsyndromic CUCLP who underwent primary lip repair from 2005 to 2011. MAIN OUTCOME MEASURE: Color pictures published in AY and Rubin's studies were used as reference pictures. Patients' photographs were cropped and rated on a 5-point scale for the vermilion border (VB), nasolabial frontal (NLF), and nasolabial profile (NLP) according to AY with/without BV assessment by Rubin's methods. Rating was performed twice by 3 oral surgeons. Intra- and inter-rater reliabilities were analyzed using weighted κ, and correlations between BV and other features were analyzed. RESULTS: Overall average assessment scores were 2.742 (0.573) with AY and 2.702 (0.489) with AY+BV methods (P = .728). Average intra-rater reliabilities were 0.605 and 0.611 and average inter-rater reliabilities were 0.525 and 0.48 with AY and AY+BV, respectively. Inter-rater reliability was the lowest for NLP. ρ scores between BV versus VB, NLF, and NLP were 0.025, 0.659, and 0.092, respectively. CONCLUSIONS: Present study demonstrates moderate intra- and inter-rater reliabilities obtained with the AY assessment method for Japanese children with CUCLP. Nasolabial profile standard ambiguity may lead to the poor reliability of AY assessment. Addition of the BV feature does not improve overall reliability.

Pilot Study of Visual and Quantitative Image Analysis of Facial Surface Asymmetry in Unilateral Complete Cleft Lip and Palate.

OBJECTIVE: To visualize and quantitatively analyze facial surface asymmetry following primary cleft lip repair in patients with unilateral cleft lip and palate (UCLP) and to compare this with noncleft controls. DESIGN: Retrospective comparative study. PATIENTS: Twenty-two patients with complete UCLP who underwent primary lip repair from 2009 to 2013 were enrolled in this study. The preserved 3-dimensional (3D) data of 23 healthy Japanese participants with the same age were used as controls. INTERVENTIONS: All patients had received primary labioplasty in accordance with Cronin triangular flap method with orbicular oris muscle reconstruction. MAIN OUTCOME MEASURES: Shadow and zebra images established from moiré images, which were reconstructed from 3D facial data using stereophotogrammetry, were bisected and reversed by the symmetry axes (the middle line of the face). The discrepancies of the gravity and density between cleft and noncleft sides in 2 regions of interest, facial and lip areas, were then calculated and compared with those of healthy participants. RESULTS: In the UCLP group, the mean discrepancies of gravity on shadow and zebra images were 1.76 ± 0.70 and 2.63 ± 1.72 pixels, respectively, in the facial area and 1.31 ± 0.36 and 3.83 ± 2.08 pixels, respectively, in the lip area. There was a significant difference in the mean discrepancies of gravity and density on zebra images in the lip area between the UCLP and control groups. CONCLUSIONS: Our image analysis of digital facial surface asymmetry in patients with UCLP provides visual and quantitative information, and it may contribute to improvements in muscle reconstruction on cleft lip repair.

Constitutive activation of p46JNK2 is indispensable for C/EBPδ induction in the initial stage of adipogenic differentiation.

Adipogenic differentiation plays a vital role in energy homeostasis and endocrine system. Several transcription factors, including peroxisome proliferator-activated receptor gamma 2 and CCAAT-enhancer-binding protein (C/EBP) α, ß, and δ, are important for the process, whereas the stage-specific intracellular signal transduction regulating the onset of adipogenesis remains enigmatic. Here, we explored the functional role of c-jun N-terminal kinases (JNKs) in adipogenic differentiation using in vitro differentiation models of 3T3-L1 cells and primary adipo-progenitor cells. JNK inactivation with either a pharmacological inhibitor or JNK2-specific siRNA suppressed adipogenic differentiation, characterized by decreased lipid droplet appearance and the down-regulation of Adiponectin, fatty acid protein 4 (Fabp4), Pparg2, and C/ebpa expressions. Conversely, increased adipogenesis was observed by the inducible overexpression of p46JNK2 (JNK2-1), whereas it was not observed by that of p54JNK2 (JNK2-2), indicating a distinct role of p46JNK2. The essential role of JNK appears restricted to the early stage of adipogenic differentiation, as JNK inhibition in the later stages did not influence adipogenesis. Indeed, JNK phosphorylation was significantly induced at the onset of adipogenic differentiation. As for the transcription factors involved in early adipogenesis, JNK inactivation significantly inhibited the induction of C/ebpd, but not C/ebpb, during the initial stage of adipogenic differentiation. JNK activation increased C/ebpd mRNA and protein expression through the induction and phosphorylation of activating transcription factor 2 (ATF2) that binds to a responsive element within the C/ebpd gene promoter region. Taken together, these data indicate that constitutive JNK activity is specifically required for the initial stage differentiation events of adipocytes.