Gene expression profiling concerning mineralization in human dental pulp cells treated with mineral trioxide aggregate.

INTRODUCTION: This study investigated changes in gene expressions related to mineralization when mineral trioxide aggregate (MTA) is applied in vitro to human dental pulp cells (HDPCs). METHODS: MTA in a Teflon tube (diameter 10 mm, height 2 mm) was applied to HDPCs. Empty tube-applied HDPCs were used as negative control. Total RNA was extracted at 6, 24, and 72 hours after MTA application for microarray analysis. The results were confirmed selectively by performing reverse transcriptase-polymerase chain reaction for genes that showed changes of more than 2-fold or less than half. RESULTS: Of the 24,546 genes, 109 genes were up-regulated more than 2-fold (eg, THBS1, VCAN, BHLHB2, FN1, COL10A1, TUFT1, and HMOX1), and 69 genes were down-regulated below 50% (eg, DCN, SOCS2, and IL8). CONCLUSIONS: These results suggest that rather than being a bio-inert material, MTA affects pulp cells in various ways. MTA appears to affect mineralization and induces slight inflammation and protective role against slight inflammation.

Localization of chondroitin sulfate proteoglycan versican in adult brain with special reference to large projection neurons.

Versican is a chondroitin sulfate proteoglycan belonging to the lectican family. Versican has two glycosaminoglycan attachment regions, named the GAG alpha and GAG beta domains, which are both regulated by alternative splicing and yield four protein isoforms. We have investigated the expression and localization of versican in the developing and adult brain by using anti-versican GAG alpha and GAG beta antibodies. Western analysis revealed that GAG alpha-reactive isoform was dominant in the adult brain. Immunohistochemical study demonstrated that GAG alpha immunoreactivity was detectable from neonatal periods to adulthood, whereas GAG beta immunoreactivity completely disappeared within 3 weeks of birth. In the adult brain, GAG alpha immunoreactivity was seen in the white matter regions and was also localized in the gray matter including somata and dendrites of cortical and hippocampal pyramidal neurons and cerebellar Purkinje cells. In contrast, GAG alpha immunoreactivity was not localized on parvalbumin-positive interneurons and cerebellar stellate cells. Furthermore, GAG alpha immunoreactivity was not co-localized with perineuronal net markers such as Wisteria floribunda agglutinin lectin and phosphacan. Thus, versican was localized on large projection neurons rather than small interneurons. To confirm the binding mechanism of versican to neurons, hyaluronan and chondroitin sulfates were enzymatically removed from brain sections before the immunolabeling of versican. These treatments had no effect on the labeling pattern of versican, suggesting that other versican-interactive molecules are involved in the binding of versican to neurons.

Versican, a major hyaluronan-binding component in the dermis, loses its hyaluronan-binding ability in solar elastosis.

Versican interacts with hyaluronan (HA) at its N-terminus and with fibrillin-1 at its C terminus. As versican in the dermis connects microfibrils to the HA-rich matrix for viscoelasticity, dermal diseases may involve destruction of these complexes. A recombinant versican protein, rVN, covering the HA binding region (HABR) of human versican and a polyclonal antibody, 6084, against rVN were prepared and characterized. Blotting analyses of skin extracts with 6084 and biotin-conjugated HA revealed that versican was a major HA-binding component in the dermis. Matrix metalloprotease-12, which is expressed in areas of solar elastosis, degraded versican and abrogated its HA-binding ability. Immunohistochemical analyses revealed that the elastic materials in solar elastosis lesions were negative for 6084, but positive for 2B1, an antibody recognizing the C-terminus of versican, indicating loss of the HABR in the aggregated elastic fibers. This loss of the HA-binding ability of versican followed by HA exclusion may be responsible for the pathological and phenotypical changes observed in solar elastosis.