Dental Follicle Cells Rescue the Regenerative Capacity of Periodontal Ligament Stem Cells in an Inflammatory Microenvironment

Aims

Periodontal ligament stem cells (PDLSCs) are one of the best candidates for periodontal regeneration. Their function could be impaired in periodontitis microenvironment. Dental follicle cells (DFCs), serving as precursor cells and mesenchymal stem cells, have intimate connection with PDLSCs. However, it is still unknown whether DFCs could provide a favorable microenvironment to improve the proliferation and differentiation capacity of PDLSCs from healthy subjects (HPDLSCs) and patients diagnosed with periodontitis (PPDLSCs).

Methods

HPDLSCs, PPDLSCs and DFCs were harvested and identified using microscopic and flow cytometric analysis. Then, the coculture systems of DFCs/HPDLSCs and DFCs/PPDLSCs were established with 0.4 µm transwell, in which all the detection indexs were obtained from HPDLSCs and PPDLSCs. The expression of stemness-associated genes was detected by real-time PCR, and the proliferation ability was assessed using colony formation and cell cycle assays. The osteogenic differentiation capacity was evaluated by real-time PCR, western blot, ALP activity, Alizarin Red S staining and calcium level analysis, while the adipogenic differentiation capacity was determined by real-time PCR and Oil Red O staining. The cell sheet formation in vitro was observed by HE staining and SEM, and the implantation effect in vivo was evaluated using HE staining and Masson’s trichrome staining.

Results

PPDLSCs had a greater proliferation capability but lower osteogenic and adipogenic potential than HPDLSCs. DFCs enhanced the proliferation and osteogenic/adipogenic differentiation of HPDLSCs and PPDLSCs to different degrees. Moreover, coculture with DFCs increased cell layers and extracellular matrix of HPDLSCs/PPDLSCs cell sheets in vitro and improved periodontal regeneration by HPDLSCs/PPDLSCs in vivo.

Conclusions

Our data suggest that the function of PPDLSCs could be damaged in the periodontitis microenvironment. DFCs appear to enhance the self-renewal and multi-differentiation capacity of both HPDLSCs and PPDLSCs, which indicates that DFCs could provide a beneficial microenvironment for periodontal regeneration using PDLSCs.     

Caenorhabditis elegans Myotubularin MTM-1 Negatively Regulates the Engulfment of Apoptotic Cells

During programmed cell death, apoptotic cells are recognized and rapidly engulfed by phagocytes. Although a number of genes have been identified that promote cell corpse engulfment, it is not well understood how phagocytosis of apoptotic cells is negatively regulated. Here we have identified Caenorhabditis elegans myotubularin MTM-1 as a negative regulator of cell corpse engulfment. Myotubularins (MTMs) constitute a large, highly conserved family of lipid phosphatases. MTM gene mutations are associated with various human diseases, but the cellular functions of MTM proteins are not clearly defined. We found that inactivation of MTM-1 caused significant reduction in cell corpses in strong loss-of-function mutants of ced-1, ced-6, ced-7, and ced-2, but not in animals deficient in the ced-5, ced-12, or ced-10 genes. In contrast, overexpression of MTM-1 resulted in accumulation of cell corpses. This effect is dependent on the lipid phosphatase activity of MTM-1. We show that loss of mtm-1 function accelerates the clearance of cell corpses by promoting their internalization. Importantly, the reduction of cell corpses caused by mtm-1 RNAi not only requires the activities of CED-5, CED-12, and CED-10, but also needs the functions of the phosphatidylinositol 3-kinases (PI3Ks) VPS-34 and PIKI-1. We found that MTM-1 localizes to the plasma membrane in several known engulfing cell types and may modulate the level of phosphatidylinositol 3-phosphate (PtdIns(3)P) in vivo. We propose that MTM-1 negatively regulates cell corpse engulfment through the CED-5/CED-12/CED-10 module by dephosphorylating PtdIns(3)P on the plasma membrane.     

The Mental Retardation Associated Protein,srGAP3 Negatively Regulates VPA-Induced Neuronal Differentiation of Neuro2A Cells

The Slit-Robo GTPase-activating proteins (srGAPs) are important multifunctional adaptor proteins involved in various aspects of neuronal development, including axon guidance, neuronal migration, neurite outgrowth, dendritic morphology and synaptic plasticity. Among them, srGAP3, also named MEGAP (Mental disorder-associated GTPase-activating protein), plays a putative role in severe mental retardation. SrGAP3 expression in ventricular zones of neurogenesis indicates its involvement in early stage of neuronal development and differentiation. Here, we show that overexpression of srGAP3 inhibits VPA (valproic acid)-induced neurite initiation and neuronal differentiation in Neuro2A neuroblastoma cells, whereas knockdown of srGAP3 facilitates the neuronal differentiation in this cell line. In contrast to the wild type, overexpression of srGAP3 harboring an artificially mutation R542A within the functionally important RhoGAP domain does not exert a visible inhibitory effect on neuronal differentiation. The endogenous srGAP3 selectively binds to activated form of Rac1 in a RhoGAP pull-down assay. We also show that constitutively active (CA) Rac1 can rescue the effect of srGAP3 on attenuating neuronal differentiation. Furthermore, change in expression and localization of endogenous srGAP3 is observed in neuronal differentiated Neuro2A cells. Together, our data suggest that srGAP3 could regulate neuronal differentiation in a Rac1-dependent manner.     

Parkin Enhances the Expression of Cyclin-dependent Kinase 6 and Negatively Regulates the Proliferation of Breast Cancer Cells

Although mutations in the parkin gene are frequently associated with familial Parkinsonism, emerging evidence suggests that parkin also plays a role in cancers as a putative tumor suppressor. Supporting this, we show here that parkin expression is dramatically reduced in several breast cancer-derived cell lines as well as in primary breast cancer tissues. Importantly, we found that ectopic parkin expression in parkin-deficient breast cancer cells mitigates their proliferation rate both in vitro and in vivo, as well as reduces the capacity of these cells to migrate. Cell cycle analysis revealed the arrestment of a significant percentage of parkin-expressing breast cancer cells at the G1-phase. However, we did not observe significant changes in the levels of the G1-associated cyclin D1 and E. On the other hand, the level of cyclin-dependent kinase 6 (CDK6) is dramatically and selectively elevated in parkin-expressing breast cancer cells, the extent of which correlates well with the expression of parkin. Interestingly, a recent study demonstrated that CDK6 restrains the proliferation of breast cancer cells. Taken together, our results support a negative role for parkin in tumorigenesis and provide a potential mechanism by which parkin exerts its suppressing effects on breast cancer cell proliferation.     

658 nm低能量激光照射对人牙周膜细胞生物学效应的影响

目的:探讨658 nm低能量激光照射对人牙周膜细胞增殖、碱性磷酸酶活性及纤维连接蛋白合成的影响.方法:改良组织块法体外培养人牙周膜细胞.通过658 nm激光照射人牙周膜细胞,观察能量密度为1.86 J/cm2和3.72 J/cm2激光照射后不同时间点细胞增殖效应、碱性磷酸酶活性和纤维连接蛋白的变化.结果:1.86 J/cm2和3.72 J/cm2能量密度的激光照射人牙周膜细胞,可显著促进细胞增殖效应.3.72 J/cm2能量密度的激光照射可提高人牙周膜细胞碱性磷酸酶活性;能量密度为1.86 J/cm2的激光照射人牙周膜细胞72 h后,细胞中纤维连接蛋白分泌量增加.结论:658 nm低剂量激光照射可促进人牙周膜细胞增殖;适量的低剂量激光照射人牙周膜细胞可促进其碱性磷酸酶活性及纤维连接蛋白的分泌.     

Age Associated Variations in RNA Biosynthesis by Cells of the Periodontal Ligament: An Autoradiographic Study1

The utilization of ³H-uridine (³H-U) was used to assess age related changes in RNA biosynthesis by connective tissue cells of the periodontal ligament. One hundred forty-four BNL mice from 5 to 78 weeks old were injected with 5μ Ci/gm body weight of ³H-U and killed from 15 min to 30 days later. Autoradiographs were prepared from 5μm thick, decalcified, sagittal sections of the maxillae. Labeled periodontal connective tissue cells were grain-counted (1) below epithelial attachment, (2) below crevicular epithelium, (3) above alveolar crest, and (4) at the alveolar crest. ³H-U incorporation was apparent at all time periods (15 min to 30 days). Nuclear labeling achieved peak values 1–4 hours after ³H-U injection. Cytoplasmic values were maximum at 1–2 days. Nuclear and cytoplasmic counts were similar at 8–16 hours. From 5 to 52 weeks of age, peak values were progressively reduced, curves were broadened and shifted towards later time periods. This age decline in the rate and magnitude of incorporation of ³H-U reflects changes in the cellular needs for RNA and protein synthesis necessary for matrix production and cellular maintenance. Some age changes were seen at 26 weeks of age. In the oldest animals (78 weeks), there appeared to be an arrest in the age-decline in ³H-U utilization. This was probably associated with the inflammatory changes reported in the periodontal ligament of old animals.     

Syndecan-2 Regulates the Migratory Potential of Melanoma Cells

Syndecan-2, a transmembrane heparan sulfate proteoglycan, is a critical mediator in the tumorigenesis of colon carcinoma cells. We explored the function of syndecan-2 in melanoma, one of the most invasive types of cancers, and found that the expression of this protein was elevated in tissue samples from both nevus and malignant human melanomas but not in melanocytes of the normal human skin tissues. Similarly, elevated syndecan-2 expression was observed in various melanoma cell lines. Overexpression of syndecan-2 enhanced migration and invasion of melanoma cells, whereas the opposite was observed when syndecan-2 levels were knocked down using small inhibitory RNAs. Syndecan-2 expression was enhanced by fibroblast growth factor-2, which is known to stimulate melanoma cell migration; however, α-melanocyte-stimulating hormone decreased syndecan-2 expression and melanoma cell migration and invasion in a melanin synthesis-independent manner. Furthermore, syndecan-2 overexpression rescued the migration defects induced by α-melanocyte-stimulating hormone treatment. Together, these data strongly suggest that syndecan-2 plays a crucial role in the migratory potential of melanoma cells.The syndecans, a family of four transmembrane cell surface heparan sulfate proteoglycans, mainly serving as a co-receptor, regulate the adhesion-dependent signal transduction of a variety of cell types, including cancer cells (1, 2). Cell adhesion receptors or co-receptors play a critical role in the neoplastic transformation of normal cells by regulating the induction of cancer-specific cellular behavior and morphology. Thus, cancer cells probably express and utilize a distinct set of syndecans in the regulation of cancer cell growth.Several reports have linked altered syndecan expression to various elements of cancer cell growth. Loss of syndecan-1 correlates with shorter survival times in patients with squamous cell carcinoma of the head, neck, and lung (3) as well as multiple myeloma (4); loss of syndecan-1 is also associated with an elevated potential for metastasis in patients with hepatocellular and colorectal carcinomas (5, 6). Previous studies have shown that syndecan-1 regulates tumor activity in pancreatic (7), gastric (8), and breast carcinomas (9). Syndecan-1 may thus play multiple roles in tumorigenic activity and perform various tissue- and/or tumor stage-specific functions (10). Syndecan-4 expression is reduced in colon carcinoma cells (11, 12) and appears to correlate with increased tumorigenic activity (e.g. cell migration and invasion (13)), implying that syndecan-4 functions as a tumor suppressor.Syndecan-2 is also known to play a crucial role in the regulation of cancer activity. Increased levels of syndecan-2 confer an invasive phenotype in lung (14) and colon cancer cells (15). Reduction in syndecan-2 expression induces cells to switch from the transformed phenotype to flattened monolayers (8) and reduces tumorigenic activity in colon adenocarcinoma and fibrosarcoma cells (8, 16). In addition, syndecan-2 is highly expressed in the microvasculature of mouse gliomas and has been shown to regulate angiogenesis in microvascular endothelial cells (17). On the other hand, an inverse correlation between syndecan-2 expression and metastatic potential has been found in Lewis lung carcinoma cell lines (6). Therefore, changes in syndecan-2 expression may directly or indirectly regulate cancer growth.Melanoma is the most aggressive malignant tumor of melanocytes. Although found predominantly in the skin, primary melanomas are also known to occur in the bowel and eye (18). Malignant melanoma is notoriously one of the most difficult cancers to treat (19). Therefore, identifying and understanding molecules that regulate the aggressive melanoma phenotype is essential for predicting the likelihood of metastasis. Interestingly, previous studies have shown that melanoma cells acquire the ability to recognize components of the extracellular matrix (ECM)² via the ectopic expression of different ECM receptors during invasion of the basement membrane (20). Indeed, invadopodia, the dynamic organelle-like structures that form actin-rich protrusions with ECM proteolytic activity, adhere to and digest collagens, laminins, and fibronectin (21). The adhesive properties of invadopodia are primarily attributed to integrins, a large family of heterodimeric transmembrane receptors composed of α and β subunits (22). For example, β₁ integrins localize within the invadopodia of melanoma cells (23), and the α₅β₁ integrins are enriched peripherally in invadopodia, where they stabilize invadopodia protrusion (24). Ectopic stimulation of α₆β₁ integrin with laminin peptides or with β₁ or α₆ integrin stimulatory antibodies increases invadopodia activity and melanoma invasiveness (23). The invasive behavior of melanoma cells can be attributed to increased cell motility caused by changes in cytoskeletal organization and altered contacts with the ECM. Thus, cell adhesion receptors may play a crucial role in the acquisition of highly migratory behavior.Syndecan-2 acts as a key regulator of cancer cells, suggesting that syndecan-2 may contribute to the aggressive phenotype and metastatic potential of melanoma. Here, we report that syndecan-2 plays a pivotal role in the migratory activity of melanoma cells.     

碱性成纤维细胞生长因子对牙周膜细胞内核心蛋白多糖基因表达的影响

利用外源性碱性成纤维细胞生长因子(Basic Fibroblast Growth Factor,bFGF)刺激体外培养的人正常牙周膜细胞.采用逆转录-聚合酶链式反应(RT-PCR)检测细胞内decorin的基因表达的变化,研究bFGF对体外培养的人牙周膜细胞内核心蛋白多糖(decorin)的作用,进一步探讨bFGF抑制Ⅰ型胶原的作用机制.发现bFGF刺激牙周膜细胞后能促进牙周膜细胞的增殖,bFGF抑制decorin的合成是bFGF促进牙周膜细胞增殖的重要调节因素之一.     

Smad2 Positively Regulates the Generation of Th17 Cells

Development of Foxp3⁺ regulatory T cells and pro-inflammatory Th17 cells from naive CD4⁺ T cells requires transforming growth factor-β (TGF-β) signaling. Although Smad4 and Smad3 have been previously shown to regulate Treg cell induction by TGF-β, they are not required in the development of Th17 cells. Thus, how TGF-β regulates Th17 cell differentiation remains unclear. In this study, we found that TGF-β-induced Foxp3 expression was significantly reduced in the absence of Smad2. More importantly, Smad2 deficiency led to reduced Th17 differentiation in vitro and in vivo. In the experimental autoimmune encephalomyelitis model, Smad2 deficiency in T cells significantly ameliorated disease severity and reduced generation of Th17 cells. Furthermore, we found that Smad2 associated with retinoid acid receptor-related orphan receptor-γt (RORγt) and enhanced RORγt-induced Th17 cell generation. These results demonstrate that Smad2 positively regulates the generation of inflammatory Th17 cells.     

Characterization of the Ca2+-Dependent Cl- Efflux in Perfused Chara Cells

The mechanism of the Ca²⁺-dependent Cl^– efflux was studiedin tonoplast-free cells, in which the intracellular chemicalcomposition can be freely controlled. Tonoplast-free cells wereprepared by perfusing the cell interior of internodal cellsof Chara corallina with a medium that contained EGTA. The Ca²⁺-inducedCl^– efflux was measured together with the membrane potentialduring continuous intracellular perfusion. The dependenciesof Cl^– efflux and the membrane potential on the intracellularCa²⁺ or Cl^– concentrations were analyzed. When perfusionwas started with medium that contained Ca²⁺ ions, Cl^–efflux and membrane depolarization were induced. The amountof Cl^– efflux varied considerably among individual cells.The rate of efflux decreased exponentially but a residual effluxremained detectable. The Cl^– efflux was induced at concentrationsof Ca²⁺ ions above 1 µM and reached a maximum at 1 mM.By contrast, the membrane depolarization reached a maximum atabout 10 µM Ca²⁺. The rate of Cl^– efflux increasedlinearly with logarithmic increases in the intracellular Cl^–concentrations. These findings suggest that more than two kindsof Ca²⁺-dependent Cl^– channel might be present in theplasma membrane. Addition of ATP or its removal from the perfusion medium didnot affect the Ca²⁺-dependent Cl^– efflux. Calmodulin antagonistsslightly inhibited the Ca²⁺-dependent Cl^– efflux. ¹Present address: Biological Laboratory, Hitotsubashi University,Naka 2-1, Kunitachi, Tokyo, 186 Japan.