Collagen induction of immune cells in the mammary glands during pregnancy.

The mammary glands are dynamic tissues affected by pregnancy-related hormones during the pregnancy-lactation cycle. Collagen production and its dynamics are essential to the remodeling of the mammary glands. Alterations of the mammary microenvironment and stromal cells during the pregnancy-lactation cycle are important for understanding the physiology of the mammary glands and the development of breast tumors. In this study, we performed an evaluation of collagen dynamics in the mammary fat pad during the pregnancy-lactation cycle. Reanalysis of single-cell RNA-sequencing (scRNA-Seq) data showed the ectopic collagen expression in the immune cells and cell-cell interactions for collagens with single-cell resolution. The scRNA-Seq data showed that type I and type III collagen were produced not only by stromal fibroblasts but also by lymphoid and myeloid cell types in the pregnancy phase. Furthermore, the total cell-cell interaction score for collagen interactions was dramatically increased in the pregnancy tissue. The data presented in this study provide evidence that immune cells contribute, at least in part, to mammary collagen dynamics. Our findings suggest that immune cells, including lymphoid and myeloid cells, might be supportive members of the extracellular matrix orchestration in the pregnancy-lactation cycle of the mammary glands.NEW & NOTEWORTHY Our study evaluated mammary gland collagen dynamics during the pregnancy-lactation cycle using single-cell RNA-sequencing data. We found ectopic collagen expression in immune cells and an increase in collagen interactions during pregnancy. Type I and type III collagen were produced by lymphoid, myeloid, and stromal fibroblast cells during pregnancy. These findings suggest that immune cells, including lymphoid and myeloid cells, play a crucial role in supporting the extracellular matrix in mammary glands during pregnancy-lactation cycles.

Supersulfides support bone growth by promoting chondrocyte proliferation in the growth plates.

OBJECTIVES: While chondrocytes have mitochondria, they receive little O2 from the bloodstream. Sulfur respiration, an essential energy production system in mitochondria, uses supersulfides instead of O2. Supersulfides are inorganic and organic sulfides with catenated sulfur atoms and are primarily produced by cysteinyl tRNA synthetase-2 (CARS2). Here, we investigated the role of supersulfides in chondrocyte proliferation and bone growth driven by growth plate chondrocyte proliferation. METHODS: We examined the effects of NaHS, an HS-/H2S donor, and cystine, the cellular source of cysteine, on the proliferation of mouse primary chondrocytes and growth of embryonic mouse tibia in vitro. We also examined the effect of RNA interference acting on the Cars2 gene on chondrocyte proliferation in the presence of cystine. RESULTS: NaHS (30 µmol/L) enhanced tibia longitudinal growth in vitro with expansion of the proliferating zone of their growth plates. While NaHS (30 µmol/L) also promoted chondrocyte proliferation only under normoxic conditions (20 % O2), cystine (0.5 mmol/L) promoted it under both normoxic and hypoxic (2 % O2) conditions. Cars2 gene knockdown abrogated the ability of cystine (0.5 mmol/L) to promote chondrocyte proliferation under normoxic conditions, indicating that supersulfides produced by CARS2 were responsible for the cystine-dependent promotion of bone growth. CONCLUSIONS: The presented results indicate that supersulfides play a vital role in bone growth achieved by chondrocyte proliferation in the growth plates driven by sulfur respiration.

M2 macrophage-derived cathepsin S promotes peripheral nerve regeneration via fibroblast-Schwann cell-signaling relay.

BACKGROUND: Although peripheral nerves have an intrinsic self-repair capacity following damage, functional recovery is limited in patients. It is a well-established fact that macrophages accumulate at the site of injury. Numerous studies indicate that the phenotypic shift from M1 macrophage to M2 macrophage plays a crucial role in the process of axon regeneration. This polarity change is observed exclusively in peripheral macrophages but not in microglia and CNS macrophages. However, the molecular basis of axonal regeneration by M2 macrophage is not yet fully understood. Herein, we aimed to identify the M2 macrophage-derived axon regeneration factor. METHODS: We established a peripheral nerve injury model by transection of the inferior alveolar nerve (IANX) in Sprague-Dawley rats. Transcriptome analysis was performed on the injured nerve. Recovery from sensory deficits in the mandibular region and histological reconnection of IAN after IANX were assessed in rats with macrophage depletion by clodronate. We investigated the effects of adoptive transfer of M2 macrophages or M2-derived cathepsin S (CTSS) on the sensory deficit. CTSS initiating signaling was explored by western blot analysis in IANX rats and immunohistochemistry in co-culture of primary fibroblasts and Schwann cells (SCs). RESULTS: Transcriptome analysis revealed that CTSS, a macrophage-selective lysosomal protease, was upregulated in the IAN after its injury. Spontaneous but partial recovery from a sensory deficit in the mandibular region after IANX was abrogated by macrophage ablation at the injured site. In addition, a robust induction of c-Jun, a marker of the repair-supportive phenotype of SCs, after IANX was abolished by macrophage ablation. As in transcriptome analysis, CTSS was upregulated at the injured IAN than in the intact IAN. Endogenous recovery from hypoesthesia was facilitated by supplementation of CTSS but delayed by pharmacological inhibition or genetic silencing of CTSS at the injured site. Adoptive transfer of M2-polarized macrophages at this site facilitated sensory recovery dependent on CTSS in macrophages. Post-IANX, CTSS caused the cleavage of Ephrin-B2 in fibroblasts, which, in turn, bound EphB2 in SCs. CTSS-induced Ephrin-B2 cleavage was also observed in human sensory nerves. Inhibition of CTSS-induced Ephrin-B2 signaling suppressed c-Jun induction in SCs and sensory recovery. CONCLUSIONS: These results suggest that M2 macrophage-derived CTSS contributes to axon regeneration by activating SCs via Ephrin-B2 shedding from fibroblasts.

Metagenomic analysis of oral plaques and aortic valve tissues reveals oral bacteria associated with aortic stenosis.

OBJECTIVES: Bacteria derived from the oral cavity enter the bloodstream and cause the onset of various systemic diseases, including heart valve disease. However, information on the oral bacteria involved in aortic stenosis is limited. MATERIALS AND METHODS: We comprehensively analyzed the microbiota in aortic valve tissues collected from aortic stenosis patients using metagenomic sequencing and investigated the relationships between the valve microbiota, the oral microbiota, and oral cavity conditions. RESULTS: Metagenomic analysis revealed the presence of 629 bacterial species in five oral plaques and 15 aortic valve clinical specimens. Patients were classified into two groups (A and B) according to their aortic valve microbiota composition using principal coordinate analysis. Examination of the oral conditions of the patients showed no difference in the decayed/missing/filled teeth index. Bacteria in group B tend to be associated with severe disease, and the number of bacteria on the dorsum of the tongue and the positive rate of bleeding during probing were significantly higher in this group than in group A. The pathophysiology of aortic stenosis may be related to the presence of oral bacteria such as Streptococcus oralis and Streptococcus sanguinis following bacteremia. CONCLUSIONS: Systemic inflammation in severe periodontitis may be driven by the oral microbiota, supporting the indirect (inflammatory) association between oral bacteria and aortic stenosis. CLINICAL RELEVANCE: Appropriate oral hygiene management may contribute to the prevention and treatment of aortic stenosis.

Suprabasin enhances the invasion, migration, and angiogenic ability of oral squamous cell carcinoma cells under hypoxic conditions.

Suprabasin (SBSN) is a secreted protein that is isolated as a novel gene expressed in differentiated keratinocytes in mice and humans. It induces various cellular processes such as proliferation, invasion, metastasis, migration, angiogenesis, apoptosis, therapy and immune resistance. The role of SBSN was investigated in oral squamous cell carcinoma (OSCC) under hypoxic conditions using the SAS, HSC­3, and HSC­4 cell lines. Hypoxia induced SBSN mRNA and protein expression in OSCC cells and normal human epidermal keratinocytes (NHEKs), and this was most prominent in SAS cells. The function of SBSN in SAS cells was analyzed using 3­(4,5­dimethylthiazol­2­yl)­2,5­diphenyltetrazolium bromide (MTT); 5­bromo­2'­deoxyuridine (BrdU); cell cycle, caspase 3/7, invasion, migration, and tube formation assays; and gelatin zymography. Overexpression of SBSN decreased MTT activity, but the results of BrdU and cell cycle assays indicated upregulation of cell proliferation. Western blot analysis for cyclin­related proteins indicated involvement of cyclin pathways. However, SBSN did not strongly suppress apoptosis and autophagy, as revealed by caspase 3/7 assay and western blotting for p62 and LC3. Additionally, SBSN increased cell invasion more under hypoxia than under normoxia, and this resulted from increased cell migration, not from matrix metalloprotease activity or epithelial­mesenchymal transition. Furthermore, SBSN induced angiogenesis more strongly under hypoxia than under normoxia. Analysis using reverse transcription­quantitative PCR showed that vascular endothelial growth factor (VEGF) mRNA was not altered by the knockdown or overexpression of SBSN VEGF, suggesting that VEGF is not located downstream of SBSN. These results demonstrated the importance of SBSN in the maintenance of survival and proliferation, invasion and angiogenesis of OSCC cells under hypoxia.

Knee meniscus regeneration using autogenous injection of uncultured adipose tissue-derived regenerative cells.

Introduction: The low healing potential of mature menisci necessitates traditional surgical removal (meniscectomy) to eliminate acute or chronic degenerative tears. However, removal of meniscal tissue is main factor causing osteoarthritis. Adipose tissue-derived regenerative cells (ADRCs), a heterogeneous cell population that includes multipotent adipose-derived stem cells and other progenitor cells, were easily isolated in large amounts from autologous adipose tissue, and same-day processing without culture or expansion was possible. This study investigated the regenerative potential of autologous ADRCs for use in meniscus defects. Methods: In 10- to 12-week-old male SD rat partial meniscectomy model, an atelocollagen sponge scaffold without or with ADRCs (5.0 × 105 cells) was injected into each meniscus defect. Reconstructed menisci were subjected to histologic, and dynamic mechanical analyses. Results: After 12 weeks, areas of regenerated meniscal tissue in the atelocollagen sponge scaffold in rats with ADRCs (64.54 ± 0.52%, P < 0.05, n = 10) were larger than in those without injection (57.96 ± 0.45%). ADRCs were shown capable of differentiating chondrocyte-like cells and meniscal tissue components such as type II collagen. Higher elastic moduli and lower fluid permeability of regenerated meniscal tissue demonstrated a favorable structure-function relationship required for native menisci, most likely in association with micron-scale porosity, with the lowest level for tissue integrity possibly reproducible. Conclusions: This is the first report of meniscus regeneration induced by injection of ADRCs. The results indicate that ADRCs will be useful in future clinical cell-based therapy strategies, including as a cell source for reconstruction of damaged knee menisci.

Human induced pluripotent stem cell-derived salivary gland organoids model SARS-CoV-2 infection and replication.

Salivary glands act as virus reservoirs in various infectious diseases and have been reported to be targeted by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the mechanisms underlying infection and replication in salivary glands are still enigmatic due to the lack of proper in vitro models. Here, we show that human induced salivary glands (hiSGs) generated from human induced pluripotent stem cells can be infected with SARS-CoV-2. The hiSGs exhibit properties similar to those of embryonic salivary glands and are a valuable tool for the functional analysis of genes during development. Orthotopically transplanted hiSGs can be engrafted at a recipient site in mice and show a mature phenotype. In addition, we confirm SARS-CoV-2 infection and replication in hiSGs. SARS-CoV-2 derived from saliva in asymptomatic individuals may participate in the spread of the virus. hiSGs may be a promising model for investigating the role of salivary glands as a virus reservoir.

Primary failure of tooth eruption: Etiology and management.

Primary failure of eruption (PFE) is a rare disorder defined as incomplete tooth eruption despite the presence of a clear eruption pathway. PFE is known to be caused by rare variants in the parathyroid hormone 1 receptor gene (PTH1R). Although several PTH1R variants have been reported, the etiology of PFE remains unclear. However, important studies that help elucidate the pathology of PFE have recently been published. The purpose of this review is to summarize current treatment options, clinical symptoms or phenotypes for diagnosis, genetic information including solid evidence in mouse disease models and disease-specific induced pluripotent stem cells, thus approaching the etiology of PFE from the perspective of the latest research.

Low oxygen tension suppresses the death of chondrocyte-like ATDC5 cells induced by interleukin-1ß.

The articular cartilage is an avascular tissue, and oxygen tensions in its superficial and deeper zones are estimated to be 6% and 1%. Degeneration of the articular cartilage begins from the surface zone in osteoarthritis. We previously reported that monocarboxylate transporter-1, a transmembrane transporter for monocarboxylates, played an essential role in the interleukin-1ß-induced expression of NADPH oxidase-2, a reactive oxygen species-producing enzyme, and reactive oxygen species-dependent death of mouse chondrogenic ATDC5 cells cultured in a normal condition (20% oxygen). Here, we investigated the effect of oxygen tension on interleukin-1ß-induced events described above in ATDC5 cells. Interleukin-1ß induced the death of ATDC5 cells under 20% and 6% oxygen but did not under 2% and 1% oxygen. Interleukin-1ß induced Mct1 (monocarboxylate transporter-1 gene) and Nox2 (NADPH oxidase-2 gene) mRNAs' expression under 20% oxygen in 24 h, respectively, but not under 2% oxygen. On the other hand, a 24-h incubation with interleukin-1ß upregulated the expression of Nos2 (inducible nitric oxide synthase gene) mRNA irrespective of oxygen tension. Furthermore, inhibition of I-κB kinase suppressed the interleukin-1ß-induced expression of Mct1 mRNA in the cells cultured under 20% and 2% oxygen, indicating NF-κB plays an essential role in the induction of the Mct1 gene expression. The results suggest that interleukin-1ß induces monocarboxylate transporter-1 in an oxygen tension-dependent manner required for cell death in ATDC5 cells. These results might explain some part of the degenerative process of the articular cartilage, which begins from its superficial zone in the pathogenesis of osteoarthritis.

Delayed Epistaxis which Was Developed after Orthognathic Surgery with Le Fort I Osteotomy and Managed by Endoscopic Cauterization.

A case of delayed epistaxis from the mucosa behind the right side of the inferior nasal mucosa 11 days after orthognathic surgery by Le Fort I osteotomy is presented. The patient was a 31-year-old man who underwent orthognathic surgery under general anesthesia. No abnormal findings were found during or after the operation. The patient was discharged from the hospital 10 days postoperatively. However, bleeding from the right nasal cavity occurred suddenly on the night after discharge, and he presented to our hospital again. The epistaxis was stopped once by nasal packing containing 0.001% epinephrine and systemic infusion of carbazochrome sulfonic acid and tranexamic acid. However, when the nasal packing was removed the next day, right nasal epistaxis was observed again. Curvature of the nasal septum and thickening of the inferior turbinate mucosa were seen on inspection; although, no active bleeding point was identified. Decreased nasal mucosa thickening and bleeding were observed after nasal packing containing 0.02% epinephrine. When the inside of the nasal cavity was observed endoscopically, an approximately 2 mm laceration was found in the mucosa behind the side wall of the right inferior nasal mucosa, and bleeding from the same part was confirmed. After endoscopic cauterization for hemostasis of the nasal mucosa, no rebleeding was observed. Although delayed epistaxis after Le Fort I osteotomy are often performed CT angiography to confirm the bleeding site, endoscopic cauterization would be primarily useful because of less invasiveness.

Extracellular acidification augments sclerostin and osteoprotegerin production by Ocy454 mouse osteocytes.

Osteocytes sense the microenvironmental stimuli, including mechanical stress, and regulate bone resorption by osteoclasts and bone formation by osteoblasts. Diabetes and cancer metastasis to bone raise l-lactic acid in the bone tissue, causing acidification. Here, we investigated the effects of l-lactic acid and extracellular acidification on the function of mouse Ocy454 osteocytes. L- and d-lactic acid with low chiral selectivity and acidification of the medium raised the production of sclerostin and osteoprotegerin by Ocy454 cells. The mRNA expression of their genes increased after either treatment of L- and d-lactic acid or acidification of the medium. Furthermore, the conditioned medium of Ocy454 cells cultured in an acidic environment suppressed the induction of alkaline phosphatase activity in MC3T3-E1 cells, which was recovered by the anti-sclerostin antibody. While it is reported that HDAC5 inhibits the transcription of the sclerostin gene, extracellular acidification reduced the nuclear localization of HDAC5 in Ocy454 cells. While calmodulin kinase II (CaMKII) is known to phosphorylate and induce extranuclear translocation of HDAC5, KN-62, an inhibitor of CaMKII lowered the expression of the sclerostin gene in Ocy454 cells. Collectively, extracellular acidification is a microenvironmental factor that modulates osteocyte functions.

Impact of Non-Coding RNAs on Chemotherapeutic Resistance in Oral Cancer.

Drug resistance in oral cancer is one of the major problems in oral cancer therapy because therapeutic failure directly results in tumor recurrence and eventually in metastasis. Accumulating evidence has demonstrated the involvement of non-coding RNAs (ncRNAs), such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), in processes related to the development of drug resistance. A number of studies have shown that ncRNAs modulate gene expression at the transcriptional or translational level and regulate biological processes, such as epithelial-to-mesenchymal transition, apoptosis, DNA repair and drug efflux, which are tightly associated with drug resistance acquisition in many types of cancer. Interestingly, these ncRNAs are commonly detected in extracellular vesicles (EVs) and are known to be delivered into surrounding cells. This intercellular communication via EVs is currently considered to be important for acquired drug resistance. Here, we review the recent advances in the study of drug resistance in oral cancer by mainly focusing on the function of ncRNAs, since an increasing number of studies have suggested that ncRNAs could be therapeutic targets as well as biomarkers for cancer diagnosis.

Induction of salivary gland-like cells from epithelial tissues transdifferentiated from mouse embryonic fibroblasts.

Salivary gland hypofunction due to radiation therapy for head and neck cancer or Sjögren syndrome may cause various oral diseases, which can lead to a decline in the quality of life. Cell therapy using salivary gland stem cells is a promising method for restoring hypofunction. Herein, we show that salivary gland-like cells can be induced from epithelial tissues that were transdifferentiated from mouse embryonic fibroblasts (MEFs). We introduced four genes, Dnp63a, Tfap2a, Grhl2, and Myc (PTMG) that are known to transdifferentiate fibroblasts into oral mucosa-like epithelium in vivo into MEFs. MEFs overexpressing these genes showed epithelial cell characteristics, such as cobblestone appearance and E-cadherin positivity, and formed oral epithelial-like tissue under air-liquid interface culture conditions. The epithelial sheet detached from the culture dish was infected with adenoviruses encoding Sox9 and Foxc1, which we previously identified as essential factors to induce salivary gland formation. The cells detached from the cell sheet formed spheres 10 days after infection and showed a branching morphology. The spheres expressed genes encoding basal/myoepithelial markers, cytokeratin 5, cytokeratin 14, acinar cell marker, aquaporin 5, and the myoepithelial marker α-smooth muscle actin. The dissociated cells of these primary spheres had the ability to form secondary spheres. Taken together, our results provide a new strategy for cell therapy of salivary glands and hold implications in treating patients with dry mouth.

Combinational Anti-tumor Effects of Chemicals from Paeonia lutea Leaf Extract in Oral Squamous Cell Carcinoma Cells.

AIM: We identified chemical components that exhibited antitumor activity against oral squamous cell carcinoma (OSCC) cells and examined their effective concentrations and additive and/or synergistic effects in combinational usage on the proliferation, apoptosis and cell cycle of OSCC cells. MATERIALS AND METHODS: Using high-performance liquid chromatography, nuclear magnetic resonance spectroscopy and electrospray ionization-mass spectrometry, we identified the main chemical components of the methanol extracts from Paeonia lutea. We investigated the pharmaceutical effects of those components on the proliferation, apoptosis, and cell cycle of an OSCC cell line, SAS, using the tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and caspase assays, as well as flow cytometry cell cycle analysis. We also examined the effects of those components on the mitogen-activated protein kinase signal transduction pathway by western blotting. Finally, the effects on normal human epidermal keratinocyte cells were also examined in similar experiments. RESULTS: Three chemicals have been identified in P. lutea leaves using high performance liquid chromatography: gallic acid methyl ester (GAME), pentagalloyl glucose (PGG) and paeoniflorin (PF). Both GAME and PGG significantly suppressed cell proliferation, and their combined effects were synergistic, while the effect of PF was minimal. However, those chemicals did not induce apoptosis. Cell cycle and western blotting analysis showed that the suppressive effects on cell proliferation resulted from G2 arrest and the suppression of phosphorylation of Akt/PKB. No effect was identified on normal human epidermal keratinocyte cells. CONCLUSION: These results indicate that GAME and PGG are the main chemical components of P. lutea leaves that have potential anti-cancer therapeutic effects.

Long-Term Follow-Up of SAPHO Syndrome for 15 Years Led to a Diagnosis of Temporomandibular Joint Pain and Trismus.

Synovitis, acne, pustulosis, hyperostosis, and osteitis (SAPHO) syndrome is a systemic disease with symptoms of pustular skin disease and sterile osteoarticular lesions. This disease rarely involves the temporomandibular joint (TMJ). Although it is a disease with a good long-term prognosis, its treatment remains challenging. We describe a case with long-term follow-up of SAPHO syndrome for 15 years in which TMJ pain and trismus led to the diagnosis. A 30-year-old woman with TMJ pain and trismus was referred to our department. Her medical history included palmoplantar pustulosis. Sterile inflammation in the left TMJ and diffuse sclerosing osteomyelitis of the mandible were observed. Thus, she was diagnosed with SAPHO syndrome. The symptoms of severe TMJ pain, trismus, and left cheek swelling presented three times in the 15 years. Symptomatic treatment with nonsteroidal anti-inflammatory drugs, antibiotics, corticosteroids, and bisphosphonates was administered several times. There has been no relapse of symptoms over the past nine years. The patient must be continuously kept under observation to look for the relapse of symptoms.

Phorbol-12-myristate 13-acetate inhibits Nephronectin gene expression via Protein kinase C alpha and c-Jun/c-Fos transcription factors.

Nephronectin (Npnt) is an extracellular matrix protein and ligand of integrin α8ß1 known to promote differentiation of osteoblasts. A search for factors that regulate Npnt gene expression in osteoblasts revealed that phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), had a strong effect to suppress that expression. Research was then conducted to elucidate the signaling pathway responsible for regulation of Npnt gene expression by PMA in osteoblasts. Treatment of MC3T3-E1 cells with PMA suppressed cell differentiation and Npnt gene expression. Effects were noted at a low concentration of PMA, and were time- and dose-dependent. Furthermore, treatment with the PKC signal inhibitor Gö6983 inhibited down-regulation of Npnt expression, while transfection with small interfering RNA (siRNA) of PKCα, c-Jun, and c-Fos suppressed that down-regulation. The present results suggest regulation of Npnt gene expression via the PKCα and c-Jun/c-Fos pathway.

Aging-associated stem/progenitor cell dysfunction in the salivary glands of mice.

Although stem cell aging leads to a decline in tissue homeostasis and regenerative capacity, it remains unclear whether salivary gland stem cell function changes during this process. However, the salivary glands are gradually replaced by connective tissue during aging. Here, we show a decline in the stem cell ability of CD133-positive stem/progenitor cells in the salivary glands of aged mice. The CD133-positive cells were isolated from young, adult, and aged mice. The number of CD133-positive cells was significantly decreased in aged mice. They also showed a lower sphere formation capacity compared to young and adult mice. RNA sequencing revealed that CD133-positive cells in aged mice exhibited lower gene expression of several aging-related genes, including FoxO3a, than those in young and adult mice. Salivary gland cells infected with a recombinant lentivirus encoding the FoxO3a gene showed a reduction in oxidative stress induced by hydrogen peroxide compared with those infected with a control virus. Thus, FoxO3a may inhibit stem cell aging via oxidative stress.

Autophagy Prevents Osteocyte Cell Death under Hypoxic Conditions.

Hypoxia occurs under important clinical conditions such as cancers, heart disease, and ischemia. However, the relationship between hypoxia and autophagy in osteocytes is still unclear. The objective of the present study was to uncover the regulatory mechanisms that prevent regulated cell death, such as apoptosis, necrosis, and autophagy, under hypoxia. MLO-Y4 cells, a mouse osteocyte cell line, were exposed to various O2 partial pressures (PO2). Subsequently, the cells underwent apoptosis, autophagy, autophagic cell death, and/or necrosis, and thereby we designated PO2 = 2% as a representative hypoxic condition. Immunofluorescence staining showed an increase of LC3 and a decrease of p62 in MLO-Y4 cells exposed to hypoxia, indicating the induction of autophagy. We then hypothesized that ß-estradiol (E2) and vitamin D play an important role in apoptosis and autophagy of osteocytes under hypoxia. 1,25α-dihydroxyvitamin D3 (VitD) protected MLO-Y4 cells from cell death and induced autophagy. However, E2 showed little effect. Finally, Western blotting for phosphorylated mTOR and Akt was carried out in order to investigate the altered autophagy signaling pathways affected by the addition of VitD and E2. However, neither E2 nor VitD were capable of recovering the decreased phosphorylation of those factors. Our results indicated that the effects of VitD on autophagy under hypoxia were dependent on the Akt and mTOR pathways. Thus, the results of the present study showed that VitD suppresses osteocyte cell death in an mTOR pathway-dependent manner in hypoxic conditions. This suggests the potential of VitD as a therapeutic intervention for diseases in which the cell death of osteocytes mainly occurs via hypoxia.

Tumor protein D52 is upregulated in oral squamous carcinoma cells under hypoxia in a hypoxia-inducible-factor-independent manner and is involved in cell death resistance.

BACKGROUND: Tumor protein D52 (TPD52) reportedly plays an important role in the proliferation and metastasis of various cancer cells, including oral squamous cell carcinoma (OSCC) cells, and is expressed strongly at the center of the tumor, where the microenvironment is hypoxic. Thus, the present study investigated the roles of TPD52 in the survival and death of OSCC cells under hypoxia, and the relationship with hypoxia-inducible factor (HIF). We examined the expression of TPD52 in OSCC cells under hypoxic conditions and analyzed the effects of HIF on the modulation of TPD52 expression. Finally, the combinational effects of TPD52 knockdown and HIF inhibition were investigated both in vitro and in vivo. RESULTS: The mRNA and protein levels of TPD52 increased in OSCC cells under hypoxia. However, the increase was independent of HIF transcription. Importantly, the observation was due to upregulation of mRNA stability by binding of mRNA to T-cell intercellular antigen (TIA) 1 and TIA-related protein (TIAR). Simultaneous knockdown of TPD52 and inhibition of HIF significantly reduced cell viability. In addition, the in vivo tumor-xenograft experiments showed that TPD52 acts as an autophagy inhibitor caused by a decrease in p62. CONCLUSIONS: This study showed that the expression of TPD52 increases in OSCC cells under hypoxia in a HIF-independent manner and plays an important role in the proliferation and survival of the cells in concordance with HIF, suggesting that novel cancer therapeutics might be led by TPD52 suppression.