HNK1 and Sox10 are present during repair of mandibular bone defects.

Neural crest cells possess characteristics of stem cells including plasticity and ability to differentiate into various cell types. HNK1 and Sox10 are markers of neural crest cell progenitors that have been demonstrated in osteoblasts during osteogenesis of the maxilla and mandible. We investigated the presence of Sox10 and HNK1 during regeneration of mandibular bone defects. Defects were created in mandibles of rats. Samples of these defects were collected at 7, 14 and 28 days post-surgery; bone regeneration was observed during this period. Immunohistochemical analysis revealed expression of HNK1 and Sox10 in osteoblasts, osteocytes and osteogenic cells, whereas osteoclasts were unstained. HNK1 expression was increased in osteoblasts and osteocytes over time and SOX10 expression was found in osteoblasts and osteogenic cells at 7, 14 and 28 days post-surgery. HNK1 and Sox10 are present in osteoblasts, osteocytes and osteogenic cells during mandible bone regeneration.

Human periodontal ligament: a niche of neural crest stem cells.

BACKGROUND AND OBJECTIVE: The periodontal ligament is a specialized connective tissue, derived from dental follicle and originated from neural crest cells. Recently it has been suggested, based on animal models, that periodontal ligament could be a niche for neural crest stem cells. However, there is still little knowledge on this subject. The identification of neural crest adult stem cells has received much attention based on its potential in tissue regeneration. The objective of the present work was to verify the human periodontal ligament as a niche for neural crest stem cells. MATERIAL AND METHODS: Cells from human periodontal ligament were isolated from 10 teeth of seven individuals (periodontal ligament pool group) and also from four teeth of one individual (periodontal ligament single group), after enzymatic digestion. The cells were cultured in specific inductive medium. Analyses of protein and gene expression were performed through immunocytochemistry and reverse transcription-polymerase chain reaction techniques, respectively. RESULTS: Mesodermal phenotypes (adipogeneic, osteogenic and myofibroblastic) were identified after culture in inductive medium. Immunocytochemistry analyses showed the presence of the nestin marker of neural stem cells and also markers of undifferentiated neural crest cells (HNK1, p75). When cultured in inductive medium that allowed neural differentiation, the cells showed markers for beta-tubulin III, neurofilament M, peripherin, microtubule-associated protein 2 and protein zero. The results were similar between the two study groups (the periodontal ligament pool group and the periodontal ligament single group). CONCLUSION: This research provides evidence that human periodontal ligament, in addition to its mesodermal derivatives, produces neural crest-like cells. Such features suggest a recapitulation of their embryonic state. The human periodontal ligament revealed itself as a viable alternative source for possible primitive precursors to be used in stem-cell therapies.

Thyroid hormone and astroglia: endocrine control of the neural environment.

Thyroid hormones (THs) play key roles in brain development and function. The lack of THs during childhood is associated with the impairment of several neuronal connections, cognitive deficits and mental disorders. Several lines of evidence point to astrocytes as TH targets and as mediators of TH action in the central nervous system; however, the mechanisms underlying these events are still not completely known. In this review, we focus on advances in our understanding of the effects of THs on astroglial cells and the impact of these effects on neurone-astrocyte interactions. First, we discuss the signalling pathways involved in TH metabolism and the molecular mechanisms underlying TH receptor function. Then, we discuss data related to the effects of THs on astroglial cells, as well as studies regarding the generation of mutant TH receptor transgenic mice that have contributed to our understanding of TH function in brain development. We argue that astrocytes are key mediators of hormone actions on development of the cerebral cortex and cerebellum and that the identification of the molecules and pathways involved in these events might be important for determining the molecular-level basis of the neural deficits associated with endocrine diseases.

Thyroid hormone induces protein secretion and morphological changes in astroglial cells with an increase in expression of glial fibrillary acidic protein.

Thyroid hormone (T3) induces in vitro differentiation of astrocytes from the developing rat brain. T3 treatment induced the appearance of long processes in cultured cerebral hemisphere and mesencephalon astrocytes from embryonic and newborn rats. T3 treatment also produced a change in the morphology of cultured cerebellar astrocytes from 10-day-old rats, but not in cerebellar astrocytes from newborn rats. An increased expression of glial fibrillary acidic protein (GFAP) was also seen in the T3-treated newborn cerebral hemisphere and mesencephalic astrocytes. The morphological changes were induced earlier when the astrocytes were treated with conditioned medium (CM) obtained from cultures previously exposed to T3. Our results show that astrocytes from the developing rat brain are not homogeneous in their responsiveness to T3. Furthermore, the fact that CM produces a response similar to that obtained with T3 treatment but in less time, suggests that T3 might induce the secretion of factors by cultured astrocytes. These factors might, by an autocrine/paracrine effect, induce the expression of GFAP and differentiation in developing brain astrocytes.

T3 affects cerebellar astrocyte proliferation, GFAP and fibronectin organization.

Thyroid hormone T3 and conditioned medium from cerebellar T3-treated astrocytes induced proliferation in astroglial cells. In addition, T3 treatment promoted alterations in the organization of cytoskeleton (GFAP) and extracellular matrix (fibronectin) in these cerebellar astrocytes in culture. GFAP filaments that normally spread in the cytoplasm of astrocytes became organized around the cell nucleus. Fibronectin that had a punctate distribution on control cell surface, became diffused in T3-treated astrocytes. This hormone also induced growth factor(s) secretion by astrocytes. These results suggest that T3 may be an important regulator of astrocyte growth and differentiation.

Thyroid hormone deficiency alters extracellular matrix protein expression in rat brain.

The effects of thyroid hormone (T3) deficiency on extracellular matrix protein expression were analyzed in newborn rat brain. In hypothyroid animals, a marked increase in the expression of 62 kDa protein was observed in cerebral hemispheres and midbrain, while the 51.6 kDa protein was increased in cerebral hemispheres and decreased in midbrain and the 44.5 kDa protein was down regulated in both structures. On the basis of molecular weights, these proteins may be the proteoglycans cerebroglycan, glypican and N-Syndecan, respectively. In addition, hypothyroidism reduced fibronectin expression in midbrain (-59,7%), but not in cerebral hemispheres. T3 deficiency affects differently the expression of proteins in different brain regions. This may be involved in brain impairment caused by hypothyroidism.

Undersulfation of glycosaminoglycans reduces the proliferation of a leukemia cell line in vitro.

Leukemia represents the clonal expansion of an individual cell lineage of the hematopoietic system at a specific point of its maturation and development. This dysregulated expansion of cells is often accompanied by altered adherence to the bone marrow microenvironment and abnormalities in endogenous cytokine production by neoplastic cells. Proteoglycans (PGs) synthesized by neoplastic cells may interact with extracellular matrix (ECM) molecules and/or locally produced cytokines. It is believed that these events may be mediated by the glycosaminoglycan (GAG) moiety of PGs such as heparan or chondroitin sulfate, and depends on its charge. The strength of GAG-cytokine binding may be determined by the extent of sulfation of the GAG chains. The synthesis, metabolism and biological role of PGs in hematopoietic malignancies have not been clearly defined. In order to study how alterations of GAGs in leukemic cells may alter cellular behavior, we treated the murine myeloid leukemic cell line WeHi-3B with sodium chlorate. This drug reduces the sulfation of GAGs, since chlorate is a potent inhibitor of sulfate adenylyltransferase. The undersulfated GAGs produced by WeHi-3B cells were not efficient in controlling the mitotic rate of the cells, since a decrease in cell proliferation was observed in vitro. These data suggest that the complexes formed by GAGs with ECM components and/or cytokines may have an important role in the induction of leukemic cell proliferation. It is possible that the stimulatory activity elicited by this binding may be dependent upon the organization of these complexes.

Thyroid hormone regulates protein expression in C6 glioma cells.

Thyroid hormone (T3) is essential to normal brain development. Previously, we have shown that T3 induces cerebellar astrocyte proliferation. This effect is accompanied by alteration in glial fibrillary acidic protein (GFAP) and fibronectin organization. In the present study, we report that the C6 glioma cell line, which expresses GFAP and is classified as an undifferentiated astrocytic cell type, is a target for T3 action. The C6 monolayers were treated with 50 nM T3 for 3 days, after which the cells were maintained for 2 days without medium changes. In C6 cells, T3 induced the expression of proteins of 107, 73 and 62 kDa. The hormone also up-regulated protein bands of 100 (+50%), 37 (+50%) and 25.5 kDa (+50%) and down-regulated proteins of 94 (-100%), 86.5 (-100%), 68 (-100%), 60 (-100%), 54 (-33%), 51 (-33%) and 43.5 kDa (-33%). We suggest, on the basis of molecular mass, that the 54-, 51- and 43.5-kDa proteins could be the cytoskeletal proteins vimentin, GFAP and actin, respectively. The down-regulation of these proteins may be involved in the effects of thyroid hormone on C6 differentiation.

Alterations in proteins of bone marrow extracellular matrix in undernourished mice.

The objective of the present study was to determine the effect of protein malnutrition on the glycoprotein content of bone marrow extracellular matrix (ECM). Two-month-old male Swiss mice were submitted to protein malnutrition with a low-protein diet containing 4% casein as compared to 20% casein in the control diet. When the experimental group had attained a 20% loss of their original body weight, we extracted the ECM proteins from bone marrow with PBS buffer, and analyzed ECM samples by SDS-PAGE (7.5%) and ECL Western blotting. Quantitative differences were observed between control and experimental groups. Bone marrow ECM from undernourished mice had greater amounts of extractable fibronectin (1.6-fold increase) and laminin (4.8-fold increase) when compared to the control group. These results suggest an association between fluctuations in the composition of the hematopoietic microenvironment and altered hematopoiesis observed in undernourished mice.

Thyroid hormone induces cerebellar astrocytes and C6 glioma cells to secrete mitogenic growth factors.

In this study, the effect of thyroid hormone (triiodothyronine, T(3)) on the secretion of mitogenic growth factors in astrocytes and C6 glioma cells was examined. The proliferating activity of T(3) could be due, at least in part, to the astrocyte secretion of acidic and basic fibroblast growth factor (aFGF and bFGF), tumor necrosis factor-beta, and transforming growth factor-beta. In contrast, the conditioned medium (CM) of T(3)-treated C6 cells was mitogenic to this cell line only after hyaluronidase digestion, suggesting the impairment of growth factor mitogenic activity by hyaluronic acid. Furthermore, the presence of bFGF was significantly greater in the CM of both T(3)-treated astrocytes and T(3)-treated C6 cells than in the corresponding control CM. These data show that T(3) induces cerebellar astrocytes to secrete mitogenic growth factors, predominantly bFGF, that could influence astrocyte and neuronal proliferation via autocrine and paracrine pathways.

Thyroid hormone and conditioned medium effects on astroglial cells from hypothyroid and normal rat brain: factor secretion, cell differentiation, and proliferation.

The effects of triiodothyronine (T3) on cell morphology were examined in cerebral hemisphere and cerebellar astrocyte cultures obtained from normal and hypothyroid neonatal rats. T3-treatment induced morphological changes in astrocytes from cerebral hemispheres. This morphological effect was produced earlier if astrocytes were treated with conditioned medium obtained from cerebral hemisphere astrocyte cultures previously exposed to 50 nM T3. T3 or conditioned medium-treatment produced faster morphological changes in hypothyroid rat cerebral hemisphere astrocyte monolayers. Cerebellar astrocytes from normal brain did not respond to thyroid hormone with morphological changes, but proliferated after T3-treatment. However, hypothyroid cerebellar astrocyte cultures exhibited morphological changes, differently than normal cells. We verified that T3 may induce astrocyte secretion of factor(s) that promotes morphological differentiation in cerebral hemisphere astroglial cultures and stimulates the proliferation of cerebellar astrocytes. Astrocytes obtained from hypothyroid animals were more sensitive to secreted factors than normal cells. These results emphasize the heterogeneity and the importance of glial cells to normal brain development and open new questions about thyroid hormone therapy in hypothyroidism.

Thyroid hormone regulates protein expression in C6 glioma cells

Thyroid hormone (T3) is essential to normal brain development. Previously, we have shown that T3 induces cerebellar astrocyte proliferation. This effect is accompanied by alteration in glial fibrillary acidic protein (GFAP) and fibronectin organization. In the present study, we report that the C6 glioma cell line, which expresses GFAP and is classified as an undifferentiated astrocytic cell type, is a target for T3 action. The C6 monolayers were treated with 50 nM T3 for 3 days, after which the cells were maintained for 2 days without medium changes. In C6 cells, T3 induced the expression of proteins of 107, 73 and 62 kDa. The hormone also up-regulated protein bands of 100 (+50 per cent), 37 (+50 per cent) and 25.5 kDa (+50 per cent) and down-regulated proteins of 94 (-100 per cent), 86.5 (-100 per cent), 68 (-100 per cent), 60 (-100 per cent), 54 (-33 per cent), 51 (-33 per cent) and 43.5 kDa (-33 per cent). We suggest, on the basis of molecular mass, that the 54-, 51- and 43.5-kDa proteins could be the cytoskeletal proteins vimentin, GFAP and actin, respectively. The down-regulation of these proteins may be involved in the effects of thyroid hormone on C6 differentiation.

Alterations in proteins of bone marrow extracellular matrix in undernourished mice

The objective of the present study was to determine the effect of protein malnutrition on the glycoprotein content of bone marrow extracellular matrix (ECM). Two-month-old male Swiss mice were submitted to protein malnutrition with a low-protein diet containing 4 percent casein as compared to 20 percent casein in the control diet. When the experimental group had attained a 20 percent loss of their original body weight, we extracted the ECM proteins from bone marrow with PBS buffer, and analyzed ECM samples by SDS-PAGE (7.5 percent) and ECL Western blotting. Quantitative differences were observed between control and experimental groups. Bone marrow ECM from undernourished mice had greater amounts of extractable fibronectin (1.6-fold increase) and laminin (4.8-fold increase) when compared to the control group. These results suggest an association between fluctuations in the composition of the hematopoietic microenvironment and altered hematopoiesis observed in undernourished mice

Undersulfation of glycosaminoglycans reduces the proliferation of a leukemia cell line in vitro

Leukemia represents the clonal expansion of an individual cell lineage of the hematopoietic system at a specific point of its maturation and development. This dysregulated expansion of cells in often accompanied by altered adherence to the bone marrow microenvironment and abnormalities in endogenous cytokine production by neoplastic cells. Proteoglycans (PGs) synthesized by neoplastic cells may interact with extracellular matrix (ECM) molecules and/or locally produced cytokines. It is believed that these events may be mediated by the glycosaminoglycan (GAG) moiety of PGs such as heparan or chondroitin sulfate, and depends on its charge. The strength of GAG-cytokine binding may be determined by the extent to sulfation of the GAG chains. The synthesis, metabolism and biological role of PGs in hematopoietic malignancies have not been clearly defined. In order to study how alterations of GAGs in leukemic cells may alter cellular behavior, we treated the murine myeloid leukemic cell line WeHi-3B with sodium chlorate. This drug reduces the sulfation of GAGs, since chlorate is a potent inhibitor of sulfate adenylyltransferase. The undersulfated GAGs produced by WeHi-3B cells were not efficient in controlling the mitotic rat of the cells, since a decrease in cell proliferation was observed in vitro. These data suggest that the complexes formed by GAGs with ECM components and/or cytokines may have an important role in the induction of leukemic cell proliferation. It is possible that the stimulatory activity elicited by this binding may be dependent upon the organization of these complexes.