טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentGurman Marina
SubjectThe Effects of Nuclear and Membrane-bound Estrogen Receptors
on Accelerated Skeletal Growth: Involvement of
Cellular Pathways
DepartmentDepartment of Medicine
Supervisors Dr. Gila Maor
Professor Raymond Coleman
Full Thesis textFull thesis text - English Version


Abstract

In the current study, we focused on the mechanisms of the endochondral ossification (EO) process regulated by estrogen (E2). EO takes place within a cartilaginous model- the epiphyseal growth plate (EGP) and is responsible for long bone formation. The accelerated maturation of growth plates, resulting in the closure of long bone growth and eventually to its termination, is controlled by E2 in both genders. E2 exerts its biological effects through two receptors isotypes: ERα and ERβ which act at both nuclear and membrane levels. As nuclear, E2 receptors act as transcriptional factors and as membrane receptors activate cellular pathways. The current study was aimed at elucidating the proper mechanisms that mediate E2 regulation of skeletal growth.

We employed two experimental models of EO; murine derived mandibular condyle (MC) organ culture and MC derived primary tissue culture (MCDC). The ER isotypes present in the cartilage cells were determined using immunoblotting and immunohistochemistry. The cellular localization of ER was determined using immunoprecipitation. The biological activity of the membrane receptor was determined using the cell impermeable derivative E2BSA. The effects on chondrogenesis and osteogenesis were estimated by following proliferation rate, proteoglycans expression and apoptosis factors. The involvement of ERs in cellular pathways was evaluated by following pAKT, pERK and pp38 expression, using ER specific modulators.   

To conclude our results, we proposed a mechanism of E2 regulation of skeletal growth. At early developmental stages, ERα is highly expressed and declines gradually as cartilage matures. Instead, ERβ expression increases with skeletal maturation. Therefore, apparently E2 through its ERα receptors affects merely early stages of cartilage development and through ERβ regulates late stages of cartilage maturation.

Furthermore, when comparing the results of E2 modulators with those achieved with E2 and E2BSA, we could demonstrate that many of PPT (ERα agonist) effects on cartilage cells resemble those of E2BSA, while DPN (ERb agonist) were similar to those of E2. Consequently, we conclude that E2, mainly through ERα receptors, influences early developmental stages, involving its membrane receptors in a non-genomic fashion. E2 through its ERβ receptors, however, causes a decline in chondrocytic proliferation, accelerates cell differentiation, maturation and senescence, in a genomic mechanism involving 'classical' E2 receptors pathway. Furthermore, we have demonstrated that E2, mainly through its cytoplasmic, rather then membrane receptors, managed to increase expression of the phosphorylated kinases, which are reported to be participated in E2 regulation of skeletal growth mechanism.