Objective: To ascertain the presence of tuftelin in mesenchymal stem cells (precursors of mineralizing tissues), and to determine their response to hypoxic stress.
Materials and Methods: The mesenchymal stem cell line C3H10T1/2 was subjected to hypoxic stress for 2-24 hours in a 1% oxygen hypoxic chamber. LDH assay, RT-PCR, DNA sequencing, Real-time PCR, SDS PAGE, Western blot, Tina 2.0 software, and immunohistochemistry, were used to localize and semi-quantify tuftelin's mRNA and protein expression.
Results: 24 hours of hypoxic insult caused an approximate 2.25 and 1.3 fold increase in tuftelin mRNA and protein expression, respectively. LDH assay after 2, 4, 6, 12, 24h of hypoxic insult indicated 0.16%, 1.84%, 1.47%, 7.32%, 7.15% cell death, respectively, depicting C3H10T1/2 cells to be stable. Immunohistochemistry revealed strong nuclear granule-like tuftelin staining after 24 hours of hypoxic stress, while the normoxic cells showed an overall weak cytoplasmic staining.
Conclusion: The present preliminary results show tuftelin is induced in C3H10T1/2 after 24 hours of hypoxic stress being among the late induced genes during hypoxia. In addition, the novel finding that tuftelin granules appear in the nucleus after hypoxia may be indicative of a cell survival signaling role for tuftelin during hypoxia. The present and ongoing structural and functional studies will contribute to the understanding of tuftelin's role in normal soft and biomineralizing tissues.
Funded by: BSF grant No 2003239.