Different stromal cell lines support lineage-selective differentiation of the multipotential bone marrow stem cell clone LyD9.

An interleukin 3-dependent multipotential stem cell clone, LyD9, has been shown to generate mature B lymphocytes, macrophages, and neutrophils by coculture with primary bone marrow stromal cells. We report here that coculture with the cloned stromal cell lines PA6 and ST2 can support differentiation of LyD9 cells predominantly into granulocyte/macrophage colony-stimulating factor (GM-CSF)- and granulocyte (G)-CSF-responsive cells, respectively. However, these stromal cell lines were unable to support lymphopoiesis of LyD9 cells. The GM-CSF-dependent line, L-GM, which was derived from LyD9 cells cocultured with PA6 stromal cells, could differentiate into macrophages and granulocytes in the presence of GM-CSF. The L-GM line can further differentiate predominantly into neutrophils by coculture with ST2 stromal cells. The G-CSF-dependent line, L-G, which was derived from LyD9 cells cocultured with ST2 stromal cells, differentiated into neutrophils in response to G-CSF. Although the stromal cell-supported differentiation of LyD9 cells required the direct contact between LyD9 and stromal cells, a small fraction of LyD9 cells that were pretreated with 5-azacytidine could differentiate into neutrophils and macrophages without direct contact with stromal cells. These results indicate that different stromal cell lines support lineage-selective differentiation of the LyD9 stem cell and that 5-azacytidine treatment can bypass the requirement of direct contact with stromal cells, albeit with a lower frequency.

TBX1 Regulates Chondrocyte Maturation in the Spheno-occipital Synchondrosis.

The synchondrosis in the cranial base is an important growth center for the craniofacial region. Abnormalities in the synchondroses affect the development of adjacent regions, including the craniofacial skeleton. Here, we report that the transcription factor TBX1, the candidate gene for DiGeorge syndrome, is expressed in mesoderm-derived chondrocytes and plays an essential and specific role in spheno-occipital synchondrosis development by inhibiting the expression of genes involved in chondrocyte hypertrophy and osteogenesis. In Tbx1-deficient mice, the spheno-occipital synchondrosis was completely mineralized at birth. TBX1 interacts with RUNX2, a master molecule of osteoblastogenesis and a regulator of chondrocyte maturation, and suppresses its transcriptional activity. Indeed, deleting Tbx1 triggers accelerated mineralization due to accelerated chondrocyte differentiation, which is associated with ectopic expression of downstream targets of RUNX2 in the spheno-occipital synchondrosis. These findings reveal that TBX1 acts as a regulator of chondrocyte maturation and osteogenesis during the spheno-occipital synchondrosis development. Thus, the tight regulation of endochondral ossification by TBX1 is crucial for the normal progression of chondrocyte differentiation in the spheno-occipital synchondrosis.

Common regulation of growth arrest and differentiation of osteoblasts by helix-loop-helix factors.

Cellular differentiation entails the coordination of cell cycle arrest and tissue-specific gene expression. We investigated the involvement of basic helix-loop-helix (bHLH) factors in differentiation of osteoblasts using the human osteoblastic cell line MG63. Serum starvation induced growth arrest at G1 phase, accompanied by expression of cyclin-dependent kinase inhibitor p21(WAF1/Cip1). Reporter assays with the p21 gene promoter demonstrated that the combination of E2A (E12 or E47) and coactivator CBP was responsible for p21 induction independent of p53. Twist inhibited E2A-CBP-dependent activation of the exogenous and endogenous p21 promoters. Ids similarly inhibited the exogenously transfected p21 promoter; however less antagonistic effect on the endogenous p21 promoter was observed. Twist was predominantly present in nuclei in MG63 cells growing in complete medium, while it localized mainly in the cytoplasm after serum starvation. The fibroblast growth factor receptor 3 gene (FGFR3), which generates signals leading to differentiation of osteoblasts, was found to be controlled by the same transcriptional regulation as the p21 gene. E2A and Twist influenced alkaline phosphatase expression, a consensus marker of osteoblast differentiation. Expression of E2A and FGFR3 was seen at the location of osteoblast differentiation in the calvaria of mouse embryos, implicating bHLH molecules in physiological osteoblast differentiation. These results demonstrate that a common regulatory system is involved in at least two distinct steps in osteoblastic differentiation. Our results also provide the molecular basis of Saethre-Chotzen syndrome, caused by mutations of the TWIST and FGFR3 genes.

Evidence for apoptosis induction in myofibroblasts during palatal mucoperiosteal repair.

Apoptosis is thought to be a requisite event for maintaining kinetic homeostasis within continually renewing tissues such as the oral mucosa and skin. However, no systematic study of the apoptotic process in fibroblasts in the oral mucosa following injury has been performed. In this study, we have assessed the expression of transforming growth factor-beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF), which are among the most important modulators of wound repair, during wound healing following mucoperiosteal injury in the rat palate. In addition, we have investigated fibroblast differentiation and apoptosis by immunohistochemical analysis for alpha-smooth-muscle (alpha-SM) actin or DNA strand breaks, respectively, to clarify the mechanisms of the wound healing process. TGF-beta1-positive cells were noted in the subepithelium from Day 2 to Day 14 after injury, by which time the wounds were completely reepithelialized. Strong expression of bFGF was observed, mainly in macrophages and monocytes at the injured site, from Day 10 to Day 14 after injury. TGF-beta1 and bFGF-immunostaining was significantly lower during the later phase of wound healing. In addition, the number of myofibroblasts expressing alpha-SM actin increased (peak at Day 14), and thereafter gradually decreased. In parallel, the apoptosis in myofibroblasts was prominent on Day 14. These results suggest that TGF-beta1 and bFGF may be potential stimulators of apoptosis in myofibroblasts after re-epithelialization in the palatal wound healing process. The regulation of apoptotic phenomena during wound healing may be important in scar establishment and development of pathological scarring.

Evidence for fibroblast growth factor receptors in myofibroblasts during palatal mucoperiosteal repair.

Fibroblast growth factors (FGFs) regulate cell growth and differentiation and play crucial roles in the process of tissue repair and remodelling. We have previously shown that basic FGF is widely expressed at the injured site. Since the presence of FGF receptors (FGFRs) determines cellular responsiveness, we examined the localisation of FGFR1, FGFR2 and FGFR3 expression by immunohistochemistry throughout the repair of full-thickness excisional wounds up to 28 days after wounding. Strong expression of FGFR1 was observed in the nuclei of myofibroblasts, which are characterised by alpha-smooth muscle (alpha-SM) actin expression. The weak expression of FGFR2 was also observed in the nuclei of myofibroblasts. In contrast, there was no staining for FGFR3 in fibroblasts through the wound healing process. In addition, transforming growth factor-beta1 (TGF-beta1), a potential inducer of myofibroblasts, enhanced the expression of FGFR1 and FGFR2 in the nuclei of palatal fibroblasts in vitro. These findings suggest that FGFR1 and FGFR2 in myofibroblasts may be responsible for the signal transduction of FGF during the wound healing process.

Basic fibroblast growth factor induces apoptosis in myofibroblastic cells isolated from rat palatal mucosa.

The effect of basic fibroblast growth factor (bFGF) on apoptosis in normal rat palatal fibroblasts and rat palatal scar fibroblasts was examined by the TUNEL method in order to clarify the mechanism of apoptosis induction in myofibroblasts during the scar formation process. A percentage of scar fibroblasts undergoing apoptosis was significantly higher than that of palatal fibroblasts when they were treated with bFGF succeeding to serum starvation. Palatal fibroblasts, phenotypically modulated into myofibroblasts by the pretreatment with transforming growth factor-beta 1 (TGF-beta 1), similarly showed a higher level of apoptosis induction by bFGF-treatment. TGF-beta 1 elevated protein and mRNA level of FGF receptor (FGFR) in palatal fibroblasts. Tyrosine autophosphorylation of FGFR upon stimulation by bFGF was significantly higher in scar fibroblasts than in normal palatal fibroblasts. These findings suggested that bFGF may be a potential stimulator of apoptosis in myofibroblasts during palatal scar formation and that FGFR may be responsible for this process.

Apoptosis of periodontal ligament cells induced by mechanical stress during tooth movement.

The mechanical force generated during tooth movement creates compressed and cell-free areas in the periodontal membrane. The way in which periodontal ligament cells disappear at the compressed area during tooth movement remains unclear. In the present study we examined whether periodontal ligament cells undergo apoptosis by mechanical stress during tooth movement using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end-labelling method (TUNEL). TUNEL-positive stainings of periodontal ligament cells began to appear at the compressed areas 12 h after tooth movement, and the number of those cells reached maximum at 24 h after tooth movement. Thereafter TUNEL-positive cells disappeared at 48 h, and direct and undermining bone resorption began at the same area 72 h after tooth movement. These results showed that compressed periodontal ligament cells were eliminated by apoptosis in the early phase of tooth movement.

Changes in rat submandibular gland N-acetyl-beta-glucosaminidase activity in streptozotocin-induced diabetes.

Changes were determined in the activity of submandibular gland N-acetyl-beta-glucosaminidase from streptozotocin-induced diabetic and insulin-treated rats. Most of the activity of this enzyme was localized histochemically in the ductal cells. The activities of this enzyme from both the subcellular supernatant and lysosomal fractions were increased in the diabetic group, and recovered to the level of the controls in the insulin-treated group. Although sex differences were observed in the activity of this enzyme, with the activity in the male rats of the control group being lower than that in the females, these differences disappeared in the diabetic group, suggesting that insulin may be related to the expression of androgen function. Both enzymes in the supernatant and lysosomal fractions were separated by isoelectric focusing into two enzymatic proteins with isoelectric points in the vicinities of pI 3 and pI 8. The effects of diabetes were reflected in an increase primarily in the activity in the vicinity of the pI 8 isoenzyme in the supernatant fraction, and in an increase in both isoenzymes in the lysosomal fraction. It is clear from these findings that the diabetic condition brings about an insulin-dependent increase in the activity of N-acetyl-beta-glucosaminidase in the rat submandibular gland, and imparts certain changes in the properties of the enzymatic proteins themselves.

Evidence for apoptosis signal-regulating kinase 1 in the regenerating palatal epithelium upon acute injury.

Apoptosis signal-regulating kinase 1 (ASK1), a recently identified mitogen-activated protein (MAP) kinase kinase kinase, is a key element in the mechanism of stress- and cytokine-induced apoptosis. However, pathophysiologic roles of ASK1 in vivo are poorly understood. In the present study, we analyzed the ASK1 expression in injured rat palate using an immunohistochemical approach to investigate the roles of ASK1 during the process of wound healing. In the normal rat palatal epithelium, a weak cytoplasmic staining of ASK1 was observed in keratinocytes of the prickle cell layer. After mucoperiosteal injury of the palate, ASK1 was clearly observed in the suprabasal keratinocytes surrounding the wound. ASK1 expression was most evident at Day 2 after injury in the edge of the migrating epithelium. Thereafter, the intensity of ASK1 staining decreased gradually until the re-epithelialization was completed at Day 10 to 14. A staining with the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end-labeling method identified a number of apoptotic keratinocytes in the suprabasal layers at the healing edge. Active induction of epithelial apoptosis was readily detectable from Day 5 after injury. In double-staining analysis, the temporal and spatial pattern of ASK1 expression correlated well with the appearance of apoptotic keratinocytes. p38 MAP kinase, a downstream component of ASK1, was found to be activated at the sites of ASK1 expression, suggesting that increased expression of ASK1 leads to activation of downstream MAP kinase signaling pathway in vivo. These results suggest a significant contribution of ASK1 to the epithelial apoptosis in the process of mucoepithelial wound repair.