Mouse Periodontal Ligament Research Articles

Identification of endoglin-dependent BMP-2-induced genes in the murine periodontal ligament cell line PDL-L2.

BackgroundThe periodontal ligament (PDL), connective tissue located between the cementum of teeth and alveolar bone of the mandibula, plays an important role in the maintenance and regeneration of periodontal tissues. We reported previously that endoglin was involved in the BMP-2-induced osteogenic differentiation of mouse PDL cells, which is associated with Smad-2 phosphorylation but not Smad-1/5/8 phosphorylation. In this study, to elucidate the detailed mechanism underlying the BMP-2 signalling pathway unique to PDL cells, we performed a microarray analysis to identify BMP-2-inducible genes in PDL-L2 cells, a mouse PDL-derived cell line, with or without endoglin knockdown.FindingsSixty-four genes were upregulated more than twofold by BMP-2 in PDL-L2 cells. Of these genes, 11 were endoglin-dependent, including Id4, which encodes ID4, a helix-loop-helix transcription factor closely associated with TGF-β signaling and osteoblast differentiation. The endoglin-dependent induction of ID4 by BMP-2 was also verified at a protein level.ConclusionOur findings indicate that ID4 could be a signal mediator involved in the BMP-2-induced endoglin-dependent osteogenic differentiation of PDL cells.

Dataset of microarray analysis to identify endoglin-dependent bone morphogenetic protein-2-responsive genes in the murine periodontal ligament cell line PDL-L2.

The periodontal ligament (PDL), connective tissue located between the cementum of teeth and alveolar bone of the mandibula, plays a crucial role in the maintenance and regeneration of periodontal tissues. We previously reported that endoglin was involved in the bone morphogenetic protein (BMP)-2-induced osteogenic differentiation of mouse PDL cells, which is associated with Smad-2 phosphorylation but not Smad-1/5/8 phosphorylation. Further, we found that the BMP-2-induced Smad-2 phosphorylation was, at least in part, dependent upon endoglin. In this study, to elucidate the detailed mechanism underlying the BMP-2-induced signaling pathway unique to PDL cells, we performed a cDNA microarray analysis to identify endoglin-dependent BMP-2-responsive genes in PDL-L2, a mouse PDL-derived cell line. Here we provide experimental methods and obtained dataset to correspond with our data in Gene Expression Omnibus (GEO) Datasets.

Hypoxia Promotes CEMP1 Expression and Induces Cementoblastic Differentiation of Human Dental Stem Cells in an HIF-1-Dependent Manner

Cementum covering the tooth root provides attachment for the tooth proper to the surrounding alveolar bone via non-mineralized periodontal ligament (PDL). Cementum protein 1 (CEMP1) has been shown to induce a cementoblastic phenotype in cementoblast precursors cells of PDL. Oxygen availability is a critical signal for correct development of many tissues; however, its role in tooth root and periodontium development remains poorly understood. In this study, we demonstrated that reduced oxygen tension increased CEMP1 expression, mineral deposition, and alkaline phosphatase activity in human dental stem cells such as PDL stem cells and periapical follicular stem cells. Since an oxemic state is transduced by the transcription factor, hypoxia-inducible factor-1 (HIF-1), we performed experiments to determine whether this protein was responsible for the observed changes. We noted that when HIF-1 was activated by gene introduction or chemically, CEMP1 expression and mineralization increased. In contrast, when HIF-1α was silenced, CEMP1 expression and mineralization did not increase in vitro. Furthermore, we showed for the first time that mouse tooth root and periodontium development occurs partly under hypoxic conditions, particularly at the apical part and latently at the PDL space in vivo. Desferrioxamine, an HIF-1 stimulator, enhances CEMP1 expression in the mouse PDL space, suggesting that hypoxia affects cementogenesis of PDL cells lining the surface of the developing tooth root in an HIF-1-dependent manner. These results suggest that HIF-1 activators may have the ability to stimulate regeneration of the tooth root and cementum formation.

Cooperative Effects of FGF-2 and VEGF-A in Periodontal Ligament Cells

We previously demonstrated that topical application of fibroblast growth factor (FGF)-2 enhanced periodontal tissue regeneration. Although angiogenesis is a crucial event for tissue regeneration, the mechanism(s) by which topically applied FGF-2 induces angiogenesis in periodontal tissues has not been fully clarified. In this study, we investigated whether FGF-2 could induce vascular endothelial growth factor (VEGF)-A expression in periodontal ligament (PDL) cells and whether cell-to-cell interactions between PDL cells and endothelial cells could stimulate angiogenesis. FGF-2 induced VEGF-A secretion from MPDL22 cells (mouse periodontal ligament cell line) in a dose-dependent manner. Transwell and wound-healing assays revealed that co-stimulation with FGF-2 plus VEGF-A synergistically stimulated the migration of MPDL22 cells. Interestingly, co-culture of MPDL22 cells with bEnd5 cells (mouse endothelial cell line) also stimulated VEGF-A production from MPDL22 cells and tube formation by bEnd5 cells. Furthermore, time-lapse analysis revealed that MPDL22 cells migrated close to the tube-forming bEnd5 cells, mimicking pericytes. Thus, FGF-2 induces VEGF-A expression in PDL cells and induces angiogenesis in combination with VEGF-A. Cell-to-cell interactions with PDL cells also facilitate angiogenesis.

Lipopolysaccharide‐induced indoleamine 2,3‐dioxygenase expression in the periodontal ligament

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-oxidizing enzyme with immune-inhibitory effects. The aim of this study was to investigate the expression of IDO by lipopolysaccharide (LPS), a component of gram-negative bacteria, in human periodontal ligament (PDL) cells. Human PDL cells and gingival fibroblasts (GFs) were prepared from explants of human PDLs and from gingival tissues of clinically healthy donors, respectively. Real-time RT-PCR, western blotting and the IDO enzyme assay were performed to determine the expression of IDO following LPS treatment of cells. LPS was injected into mice tail veins to evaluate the effects of LPS in vivo in the maxillary first molar. Immunofluorescence staining and histological analysis were followed to localize IDO in mouse PDL. The level of expression of IDO mRNA in primary human PDL cells after LPS treatment was increased in a dose-dependent manner, reaching a peak 8h after LPS treatment. The expression and activities of IDO protein were significantly increased in comparison with those of the control. In addition, the increased production of kynurenine in culture medium was observed 72h after LPS treatment. In the immunofluorescence findings, stronger immunoreactivities were shown in PDL than in gingival tissues in the maxillae. In accordance with the immunofluorescence findings, LPS treatment induced a strong up-regulation of IDO mRNA in human PDL cells, whereas human GFs showed only a weak response to LPS. These results clearly show that IDO was induced by LPS in primary human PDL cells, suggesting that PDL might be involved in the regulation of oral inflammatory disease.

α11β1 integrin‐mediated MMP‐13‐dependent collagen lattice contraction by fibroblasts: Evidence for integrin‐coordinated collagen proteolysis

We have previously determined that integrin α11β1 is required on mouse periodontal ligament (PDL) fibroblasts to generate the force needed for incisor eruption. As part of the phenotype of α11(-/-) mice, the incisor PDL (iPDL) is thickened, due to disturbed matrix remodeling. To determine the molecular mechanism behind the disturbed matrix dynamics in the PDL we crossed α11(-/-) mice with the Immortomouse and isolated immortalized iPDL cells. Microarray analysis of iPDL cells cultured inside a 3D collagen gel demonstrated downregulated expression of a number of genes in α11-deficient iPDL cells, including matrix metalloproteinase-13 (MMP-13) and cathepsin K. α11(-/-) iPDL cells in vitro displayed disturbed interactions with collagen I during contraction of attached and floating collagen lattices and furthermore displayed reduced MMP-13 protein expression levels. The MMP-13 specific inhibitor WAY 170523 and the Cathepsin K Inhibitor II both blocked part of the α11 integrin-mediated collagen remodeling. In summary, our data demonstrate that in iPDL fibroblasts the mechanical strain generated by α11β1 integrin regulates molecules involved in collagen matrix dynamics. The positive regulation of α11β1-dependent matrix remodeling, involving MMP-13 and cathepsin K, might also occur in other types of fibroblasts and be an important regulatory mechanism for coordinated extracellular and intracellular collagen turnover in tissue homeostasis.

Role of ferritin in the cytodifferentiation of periodontal ligament cells

This study investigated the expression and functions of ferritin, which is involved in osteoblastogenesis, in the periodontal ligament (PDL). The PDL is one of the most important tissues for maintaining the homeostasis of teeth and tooth-supporting tissues. Real-time PCR analyses of the human PDL revealed abundant expression of ferritin light polypeptide (FTL) and ferritin heavy polypeptide (FTH), which encode the highly-conserved iron storage protein, ferritin. Immunohistochemical staining demonstrated predominant expression of FTL and FTH in mouse PDL tissues in vivo. In in vitro-maintained mouse PDL cells, FTL and FTH expressions were upregulated at both the mRNA and protein levels during the course of cytodifferentiation and mineralization. Interestingly, stimulation of PDL cells with exogenous apoferritin (iron-free ferritin) increased calcified nodule formation and alkaline phosphatase activity as well as the mRNA expressions of mineralization-related genes during the course of cytodifferentiation. On the other hand, RNA interference of FTH inhibited the mineralized nodule formation of PDL cells. This is the first report to demonstrate that ferritin is predominantly expressed in PDL tissues and positively regulates the cytodifferentiation and mineralization of PDL cells.

Identification and characterization of neural crest-derived cells in adult periodontal ligament of mice

ObjectiveCells derived from the neural crest (NC) contribute to the development of several adult tissues, including tooth and periodontal tissue. Here, two transgenic lines, Wnt1-Cre/ZEG and P0-Cre/ZEG, were analysed to determine the fate and distribution of neural crest cells (NCCs) in adult mouse periodontal ligament (PDL). DesignParaffin-embedded and decalcified histology samples were prepared from Wnt1-Cre/ZEG and P0-Cre/ZEG mice that were 4-, 8-, or 12-weeks old. Expression of GFP (NC-derived cells), NC-markers (Slug, AP-2 alpha, HNK-1, p75NTR and Nestin), and mesenchymal stem cell markers (CD29 and STRO-1) were examined using immunohistochemistry. ResultsIn four-week-old Wnt1-Cre/ZEG mice, GFP(+) NC-derived cells were specifically detected in the mid-zone of PDL. The GFP(+) cells constituted 1.4% of all cells in PDL, and this percentage decreased as the mice aged. The distribution and prevalence of GFP(+) cells were comparable between Wnt1-Cre/ZEG and P0-Cre/ZEG mice. NC-marker(+) cells were expressed only in GFP(+) cells while MSC markers were detected only in GFP(−) cells. ConclusionThe prevalence and specific distribution of NC-derived cells in adult PDL of Wnt1-Cre/ZEG and P0-Cre/ZEG mouse were examined. Interestingly, various NC markers, including markers for undifferentiated NCCs, were still expressed at high levels in GFP(+) cells. These observations may indicate that labelled cells in the Wnt1-Cre/ZEG and P0-Cre/ZEG mice did not constituted all NC-derived cells, but rather an interesting subset of NC-derived cells. These findings may be useful in understanding the homeostatic character of the PDL and contribute to establishing successful periodontal tissue maintenance.

Ameloblastin regulates cell attachment and proliferation through RhoA and p27

The matrix adhesion protein ameloblastin (AMBN) is one of the unique components of the mineralizing matrix of bones and teeth. Here we focused on two types of cells expressing AMBN - mouse dental follicle cells (mDF) and mouse periodontal ligament cells (mPDL) - to decipher AMBN function in developing dental, periodontal, and bone tissues. To test AMBN function, cell culture dishes of mDF and mPDL were exposed to either full-length or C-terminal (amino acids 137-407) recombinant Ambn protein. Alternatively, cells were subjected to transient transfection using an Ambn-small hairpin (sh) RNA vector. Our cell culture studies documented that dishes coated with full-length AMBN promoted the attachment of mPDL and mDF cells as early as 1 h after seeding. In order to identify potential intermediaries that might aid the effect of AMBN on adhesion, RhoA expression levels in AMBN-coated and uncoated control dishes were assessed. These studies indicated that AMBN induced RhoA expression 4 h after seeding, especially in mPDL cells. After 4 h of culture, the cell cycle inhibitor p27 was also up-regulated. In addition, exogenous AMBN and its C-terminal fragment reduced the proliferation of mDF and mPDL. Finally, transient transfection of mDF and mPDL cells with the Ambn-shRNA vector resulted in the down-regulation of p27 in mPDL cells. Together, these data indicate that AMBN affects cell adhesion via RhoA and cell cycle progression through p27.

Osteoinductive and anti-inflammatory effect of royal jelly on periodontal ligament cells

Royal jelly (RJ) has been reported to possess several physiological and pharmacological properties such as the ability to prevent osteoporosis in rats and anti-inflammatory effects. We hypothesized that RJ could have beneficial effects on the prevention or treatment of periodontal diseases, which are chronic inflammatory diseases caused by bacterial infection that result in resorption of the tooth-supporting bone. We assessed the effect of RJ on mineralization in mouse periodontal ligament cell clone 22 (MPDL22 cells), which are of an osteogenic and cementogenic lineage. The mRNA expression of osteopontin, osteocalcin and osterix, and mineralized nodule formation were significantly enhanced in RJ-treated MPDL22 cells. In addition, we investigated the effects of RJ on the production of inflammatory cytokines from MPDL22 cells stimulated with lipopolysaccharide (LPS) of Porphyromonas gingivalis, a periodontopathic bacterium. RJ suppressed LPS-induced interleukin-6 and CXC chemokine ligand 10 production from MPDL22 cells. Furthermore, RJ suppressed the expression of CD54 in MPDL22 cells: CD54 is the adhesion molecule involved in the accumulation of leukocytes in periodontal lesions. These findings suggest that the osteoinductive and anti-inflammatory effects of RJ can provide benefits for the treatment and prevention of periodontal diseases.

Lactone form 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) stimulate the osteoblastic differentiation of mouse periodontal ligament cells via the ERK pathway

Recent studies reported that the lactone forms of 3-hydroxy- 3-methylglutaryl-coenzyme A reductase inhibitors, which are also known as statins, have a bone stimulatory effect. However, there are few reports on the effect of statins on periodontal ligament cells. This study examined the statin-induced osteoblastic differentiation of mouse periodontal ligament cells as well as its mechanism. Mouse periodontal ligament cells were cultured with lovastatin or simvastatin, and their viability was measured. The levels of alkaline phosphatase (ALP), osteocalcin, bone sialoprotein and bone morphogenetic protein-2 mRNA expression were evaluated by RT-PCR. The osteoblastic differentiation was characterized by the ALP activity and Alizarin Red-S staining for calcium deposition. The activity of the osteocalcin gene (OG2) and synthetic osteoblast-specific elements (6× OSE) promoter with statins was also measured using a luciferase assay. For the signal mechanism of statins, the ERK1/2 MAPK activity was determined by western blot analysis. A statin treatment at concentrations < 1 μM did not affect the cell viability. Lovastatin or simvastatin at 0.1 μM increased the levels of ALP, osteocalcin, bone sialoprotein and bone morphogenetic protein-2 mRNA in mouse periodontal ligament cells. In addition, the ALP activity, mineralized nodule formation and OG2 and OSE promoter activity were higher in the lovastatin- or simvastatin-treated cells than the control cells. Western blot analysis confirmed that the statins stimulated the phosphorylation of ERK1/2. Lovastatin and simvastatin may stimulate the osteoblastic differentiation of periodontal ligament cells via the ERK1/2 pathway. This suggests that the statins may be useful for regenerating periodontal hard tissue.

Fibroblast growth factor‐2 stimulates directed migration of periodontal ligament cells via PI3K/AKT signaling and CD44/hyaluronan interaction

Fibroblast growth factor-2 (FGF-2) regulates a variety of functions of the periodontal ligament (PDL) cell, which is a key player during tissue regeneration following periodontal tissue breakdown by periodontal disease. In this study, we investigated the effects of FGF-2 on the cell migration and related signaling pathways of MPDL22, a mouse PDL cell clone. FGF-2 activated the migration of MPDL22 cells and phosphorylation of phosphatidylinositol 3-kinase (PI3K) and akt. The P13K inhibitors, Wortmannin and LY294002, suppressed both cell migration and akt activation in MPDL22, suggesting that the PI3K/akt pathway is involved in FGF-2-stimulated migration of MPDL22 cells. Moreover, in response to FGF-2, MPDL22 showed increased CD44 expression, avidity to hyaluronan (HA) partly via CD44, HA production and mRNA expression of HA synthase (Has)-1, 2, and 3. However, the distribution of HA molecular mass produced by MPDL22 was not altered by FGF-2 stimulation. Treatment of transwell membrane with HA facilitated the migration of MPDL22 cells and an anti-CD44 neutralizing antibody inhibited it. Interestingly, the expression of CD44 was colocalized with HA on the migrating cells when stimulated with FGF-2. Furthermore, an anti-CD44 antibody and small interfering RNA for CD44 significantly decreased the FGF-2-induced migration of MPDL22 cells. Taken together, PI3K/akt and CD44/HA signaling pathways are responsible for FGF-2-mediated cell motility of PDL cells, suggesting that FGF-2 accelerates periodontal regeneration by regulating the cellular functions including migration, proliferation and modulation of extracellular matrix production.

Growth Differentiation Factor 5 (GDF-5) Induces Matrix Metalloproteinase 2 (MMP-2) Expression in Periodontal Ligament Cells and Modulates MMP-2 and MMP-13 Activity in Osteoblasts

Growth differentiation factor 5 (GDF-5), a member of the transforming growth factor beta (TGF-β) superfamily expressed in periodontal tissues, promotes extracellular matrix (ECM) in periodontal ligament (PDL) cells. Matrix metalloproteinases (MMP) are proteolytic enzymes that degrade ECM and are expressed in PDL cells. To date, little is known about the regulation of MMP synthesis and secretion in PDL cells. The aim of this study is to examine the effects of GDF-5 on MMP production and activity in PDL cells. GDF-5 increased both collagen type I alpha 2 (Col Iα2) and MMP-2 gene expression in cells derived from mouse PDL tissues after 3 days of culture. Because PDL cells represent a heterogeneous population, we examined gelatinolytic activity and gene expression profiles in an osteoblast cell line. After 6, 12, 24, and 48 hours of culture, GDF-5 increased both Col Iα2 and MMP gene expression in osteoblasts. GDF-5 also promoted MMP-2 activity as revealed by gelatin zymography after 7 days of culture. In the presence of the p38 MAP kinase inhibitor SB202190, on the other hand, MMP-2 activity was blocked. Taken together, these results indicate that GDF-5 may increase simultaneously the gene expression of type I collagen and MMP-2 in the osteoblast-like cells among the PDL cells and p38 MAP kinase pathway in osteoblast could involve in the regulation of MMP-2 enzyme activity induced by GDF-5.

Lymphatic distribution in the mouse periodontal ligament

Lymphatic distribution in the mouse periodontal ligament

Fibroblast growth factor‐2 regulates expression of osteopontin in periodontal ligament cells

Osteopontin is a protein found in the bone-related matrix and plays multiple regulatory roles in mineralizing and non-mineralizing tissue. In osteogenic cell-lines, the expression of osteopontin increases with the progression of differentiation, but both the expression and function of osteopontin vary with the cell type and its activation state. In this study, we examined the expression of osteopontin by clones established from mouse periodontal ligament, in response to inorganic phosphate and fibroblast growth factor (FGF)-2, which can induce periodontal tissue regeneration. The involvement of inorganic phosphate in the expression of osteopontin during the course of cell differentiation of a clone MPDL22 was confirmed by addition of foscarnet, an inorganic phosphate transport inhibitor. Although FGF-2 decreased the mRNA expression of almost every bone-related protein in MPDL22, FGF-2 upregulated the expression of osteopontin in MPDL22 at both mRNA and protein levels. Interestingly, FGF-2 enhanced the concentration of osteopontin in the culture supernatant of MPDL22, whereas inorganic phosphate did not. The FGF-2-induced osteopontin in the culture supernatant seems to be involved in cell survival activity. An immunohistochemical study showed that the FGF-2-induced osteopontin was mainly present in perinuclear matrices while the inorganic phosphate-induced osteopontin was associated with extracellular matrices in addition to perinuclear matrices. The present results indicated that FGF-2 induces unique expression of osteopontin, which may play a role different from the other bone-related proteins during the process of periodontal tissue regeneration by FGF-2.

Transcriptome database KK-Periome for periodontal ligament development: Expression profiles of the extracellular matrix genes

Specialized connective tissues such as tendon/ligament develop through a series of events that require temporal and spatial expression of numerous genes in mesenchymal progenitors. However, the genes required for tendon/ligament development have not been identified yet. To solve this problem, we made a cDNA library from periodontal ligament and sequenced 11,520 cDNA clones, as a model for investigating tendon/ligament development. The resulting sequence data was assembled to 617 expressed sequence tag (EST) clusters, and an EST database for human periodontal ligament (PDL) was constructed (designated as the KK-Periome database). In the KK-Periome database, the top 13 EST clusters were related to extracellular matrix (ECM) genes. The temporal and spatial expression patterns of these genes during mouse PDL development were examined by in situ hybridization. Among these genes, F-spondin was expressed specifically in dental follicle (DF) cells during tooth germ development, whereas tenascin-N was strongly expressed in the terminally differentiated PDL. This characteristic expression profile was confirmed by in vivo differentiation assay of human PDL (hPDL) cells in the mouse transplant. Thus, the KK-Periome database was proven to be a useful resource for PDL-derived ESTs (transcriptome), and in fact, initial evidence indicated that F-spondin and tenascin-N might serve as markers for DF and PDL, respectively.

The role of p38 MAP kinase on RANKL regulation in mouse periodontal ligament fibroblasts

The role of p38 MAP kinase on RANKL regulation in mouse periodontal ligament fibroblasts

Messenger RNA expression of periostin and Twist transiently decrease by occlusal hypofunction in mouse periodontal ligament

Periostin, which is a secreted protein that supports cell adhesion, is highly expressed in the periodontal ligament (PDL). Twist, a basic helix-loop-helix (bHLH) transcription factor and a negative regulator of osteoblast differentiation, has been found to regulate the periostin gene transcription. Since occlusal force is thought to be important in the homeostasis of the PDL, in this study we investigated the expression of periostin and Twist mRNA in the mouse periodontal tissue following removal of antagonizing teeth. Unilateral maxillary tooth extraction was performed in 3-week-old male mice to produce occlusal hypofunction of the right mandibular molars. The expressions of periostin and Twist mRNA were examined by real time-PCR and in situ hybridization at 12, 24, 72 and 168 h after the tooth extraction. The real-time PCR analysis showed that periostin and Twist mRNA significantly decreased at 24 h to 14.5 and 49.9% of those in control group, respectively. But the recovery began at 72 and 168 h, no significant difference was observed. As determined by in situ hybridization analysis, the number of periostin and Twist mRNA-expressing PDL cells showed a marked decrease at 24 h, although an increase was observed from 72 h until the distribution was almost similar to that of the control group at 168 h. These results suggested that occlusal force might have putative roles in periostin and Twist gene expression in the PDL and the changes in their expression level during hypofunction may be considered a form of adaptation to environmental changes.

Ultrastructural changes in postcapillary-sized venule morphology in aged mouse periodontal ligament

Abstract The aim of this study was to compare the postcapillary-sized venule (PCV) morphology of four young ALCA mice (35 days) and four colony-related aged mice (365 days) using the transmission electron microscope (TEM). Right and left mandibular first molar mesial roots with associated periodontal ligament (PDL) and bony socket, were used for TEM assessment. Five PCV profiles were selected at each 160µm interval, from the alveolar crest to the tooth apex. PCV profile dimensions were measured on standardised micrographs magnified x2900. Age affects were tested using multiple regression analysis. The number of PCV profiles in the tooth third of the PDL was higher in aged mice (p &lt; 0.01) and comprised predominantly apericytic vessels (p &lt; 0.001). The number of PCV profiles increased significantly (p &lt; 0.001) in aged mice in the PDL middle circumferential third halfway down the molar root. Age had no significant affect on PCV diameter. Aged PDL permeability studies are needed to investigate whether the changes in aged PCV profile number are associated with functional modification of the PDL microvasculature.

Mitogenic effects of neutrophins on a periodontal ligament cell line.

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) are three representative neurotrophins responsible for the differentiation and survival of neurons, and their high-affinity receptors are tropomyosin-receptor-kinase (TRK)A, TRKB, and TRKC, respectively. In this study, we investigated the expression of neurotrophins in a mouse periodontal ligament cell line (MPL), by reverse transcription-polymerase chain-reaction (RT-PCR) and enzyme-linked immunoabsorbent assay (ELISA). We also studied the expression of TRK receptors on MPL by immunostaining and the effects of neurotrophins on the proliferation of MPL, with a hypothesis of autocrine mechanism of neurotrophins. Each neurotrophin and TRK receptor was expressed, and neurotrophins enhanced the proliferation of MPL. These findings suggest that the MPL has functional neurotrophin receptors involved in an autocrine function of neurotrophins. The expression level of neurotrophins and TRKs showed the reverse pattern, and we propose an auto-regulatory mechanism of ligands and receptors in accordance with the level of synthesized neurotrophins.