The Research Of STIM1 Regulated Osteoblast Differentiation In The Development Of Postmenopausal Osteoporosis

Background:Osteoporosis is typically a disease of the elderly, and with population aging it has become one of the most frequent and relevant health problems in this age bracket, especially among women. The disease is characterized by low bone mineral density（BMD） and degeneration of the bone microarchitecture, which increase the bone brittleness and fracture risk and significantly reduce the quality of life of patients. BMD can be assessed with dual Xray absorptiometry（DXA）, and osteoporosis is defined by T-score. The lifetime fracture risk of a patient with osteoporosis is as high as 40%, and fractures most commonly occur in the spine, the hip, or the wrist, but other bones such as the humerus or ribs may also be involved. Osteoporosis therapies fall into two classes, anti-resorptive drugs, which slow down bone resorption or anabolic drugs, which stimulate bone formation. Calcium is required for a number of functions in the body, including neuromuscular activity, membrane function, hormone secretion, enzyme activity, coagulation of the blood and skeletal mineralization. Over 99% of the body’s calcium is stored in bone, where it provides mechanical strength to the skeleton, and serves as a mineral reservoir that can be drawn upon to maintain a normal plasma calcium. Adequate intakes of calcium are essential preventive strategies and essential parts of any therapeutic regimen for osteoporosis. Experimentation on a variety of animal models for over 80 years has clearly demonstrated that low dietary calcium, particularly in adult animals, produces osteoporosis, defined as a low quantity of normally mineralized bone. Past research has established the strong correlation between calcium homeostasis and supplementation leading to enhanced bone health in postmenopausal women. However, calcium supplementation is not without controversy and benefits on skeletal health need to be balanced against potential risks. For many decades there has been advocacy for the use of calcium supplements in the prevention and treatment of osteoporosis. However, result of recent clinical trials indicate that calcium supplementation does not significantly reduce fracture risk in postmenopausal women. In addition, calcium supplementation may cause variety of adverse effects such as cardiovascular disease, constipation and kidney stone. All these findings arose highly attention of researchers and publics. What is the exact effect of calcium supplementation on prevention of osteoporosis and fracture? Is there any chance that the Calcium channel of osteoporosis patient is dysfunction which make the Ca²⁺ failed to entry into the cells? 17β-E₂ is known for its essential role in bone mass and density maintenance, and its deficiency leads to the progression of postmenopausal osteoporosis. Research shows that 17β- E₂ could induce Ca²⁺ influx by upregulate L-type voltage-sensitive channels in rat hippocampal neurons via PI3K-Akt signal pathway. Besides, the function of SOCE could be promoted through Akt-m TOR signaling pathway. Therefore, we hypothesis that 17β- E₂ might have similar effect on other calcium channels and these channels might be abnormal due to deficiency of 17β- E₂ in postmenopausal woman, which make the Ca²⁺ failed to entry into the cells. Store-operated Ca²⁺ entry（SOCE） is an important Ca²⁺ influx pathway in many non-excitable and some excitable cells. It is regulated by the filling state of intracellular Ca²⁺ stores, notably the endoplasmic reticulum（ER）. Reduction in [Ca²⁺] ER results in activation of plasma membrane Ca²⁺ channels that mediate sustained Ca²⁺ influx which is required for many cell functions as well as refilling of Ca²⁺ stores. The Ca²⁺ release activated Ca²⁺（CRAC） channel is the best characterized SOCE channel with well-defined electrophysiological properties. In recent years, the molecular components of the CRAC channel, long mysterious, have been defined. Stromal interaction molecule-1（STIM1） is localized in the endoplasmic reticulum（ER）, senses [Ca²⁺] ER, and activates the CRAC channel upon store depletion. Recent research found that CRAC channel play an essential role in differentiation and function of osteoclasts and osteoblasts. Here we have investigated the role of STIM1 in the osteogenic differentiation of osteoblasts and postmenopausal osteoporosis, futher studies are needed to clarify potential mechanisms and provide new strategy for treatment.Aims:Identify the mechanism of STIM1 regulated osteoblasts differentiation in postmenopausal osteoporosis.Methods:1. To investigate the function of CRAC channel of primary osteoblasts derived from bone marrow mesenchymal stem cells（BMMSCs）, BMMSCs were cultured in the presence of osteogenic induction for 14 d after obtained from postmenopausal osteoporosis patients. We analyzed the Ca²⁺ concentration and CRAC channel by Fluo-3 staining before and after osteogenic induction respectively. STIM1 expression and osteogenic differentiation was further evaluated using quantitative real time PCR to compare expression of STIM1 and osteogenic markers. RNA was collected from cultures at the start of osteogenic induction and after 3, 7 and 14 days. BMMSCs were stained for alkaline phosphatase（ALP） at 7d, which is an early osteogenic differentiation marker. Mineralization was evaluated using the calcium chelating dye alizarin red.2. We knockdown one of component of CRAC channel, STIM1 in MC3T3-E1 using stable sh RNA interference. Detect the osteogenic gene markers, ALP activity and mineralization ability to identify whether STIM1 is involed in osteogenesis.3. We next investigated the effects of 17β-estradiol on STIM1. MC3T3-E1 were cultured in the presence of 17β-estradiol and 17β-estradiol inhibitor（ICI） while osteogenic induction. RT-PCR and Western Blot was used to detect the expression and m RNA transcript levels. To elucidate the underlying mechanism of estrogen on STIM1, we added inhibitor of PI3 K and m TOR to determine whether this signaling pathway was involved.4. To investigate the role of STIM1 in osteoblast differentiation and osteogenesis regulated by 17β-estradiol, we compared the osteogenic gene markers, ALP activity and mineralization ability between STIM1 knockdown and control group in the presence of 17β-estradiol.Results:1. To investigate the function of CRAC channel of primary osteoblasts derived from bone marrow mesenchymal stem cells（BMMSCs）, BMMSCs were cultured in the presence of osteogenic induction for 14 d after obtained from postmenopausal osteoporosis patients. Ca²⁺ imaging analysis showed that Ca²⁺ concentration was significantly reduced in BMMSCs of osteoporosis. After ER depletion triggered by thapsigargin（TG）, Ca²⁺ influx through CRAC channel was significantly decreased in osteoporosis before as well as after osteogenic induction compared to control group. Real Time-PCR found that STIM1 expression was decreased in BMMSCs of postmenopausal osteoporosis. Osteogenic markers expression of Alp, Collagen1, Opn, Ocn was relatively low in osteoporosis. After 14 d culture in osteogenic medium, mineralization was readily apparent. However, alizarin red staining revealed a marked reduction in postmenopausal osteoporosis. Similarly, ALP staining was significantly reduced in postmenopausal osteoporosis. These observations indicating that the function of CRAC channel and osteogenic differentiation of BMMSCs is decreased in postmenopausal osteoporosis patient. We supposed that there is a correlation between the dysfunction of CRAC channel and the decreased osteogenic differentiation of BMMSCs, and this may be a factor contributes to osteoporosis.2. Based on the findings that the function of CRAC channel and osteogenic potential is decreased in BMMSCs of postmenopausal osteoporosis, to further explore the role of CRAC channel in the osteogenic differentiation, we knock down one of component of CRAC channel, STIM1 in MC3T3-E1 using stable sh RNA interference. Real time-PCR showed that the expression of osteogenic markers were significantly reduced in STIM1 sh RNA group of whole osteogenic induction process. Statistical analysis of both ALP and Alizarin Red staining showed that knockdown of STIM1 weakened the osteogenic differentiation and mineralization of osteoblasts derived from MC3T3-E1 dramatically. All these findings showed that STIM1 is essential to osteogenic differentiation of osteoblasts.3. 17β-E₂ is known for its essential role in bone mass and density maintenance, and its deficiency leads to the progression of postmenopausal osteoporosis. We next investigated the effects of 17β-estradiol on STIM1. MC3T3-E1 were cultured in the presence of 17β-estradiol and 17β-estradiol inhibitor（ICI） while osteogenic induction. The results of Real time-PCR and Western blot found that 17β- E₂ can promotes the expression of STIM1. Fluo-3 staining showed stronger Ca²⁺ influx in the presence of 17β- E₂, indicating the function of CRAC have been improved. To elucidate the underlying mechanism of estrogen on STIM1, we added inhibitor of PI3 K and m TOR to identify the potential signaling pathway. Electron micrographs illustrating ultrastructural characteristics of osteoblasts. Results shows that expand of ER was promotes by 17β- E₂, and this effect was offset by inhibitors of 17β- E₂ and PI3K-m TOR signaling pathway. Ca²⁺ imaging analysis showed that Ca²⁺ influx was reduced, Real time-PCR and Western blot found that these inhibitors have a negative effect on expression of STIM1. All these results indicated that 17β- E₂ might promotes the STIM1 expression via the PI3K-m TOR signaling pathway.4. To investigate the role of STIM1 in osteoblast differentiation and osteogenic regulated by 17β- E₂, we compared the osteogenic gene markers between STIM1 knockdown and control group in the presence of 17β- E₂. Real time-PCR showed that these markers were significantly reduced in STIM1 knockdown group in the presence of 17β- E₂. The results of ALP and Alizarin red staining were consistent with Real time-PCR. These observations suggest that STIM1 play an essential role in osteogenic differentiation of osteoblasts regulated by estrogen.Conclusion:In summary, we found that the expression of STIM1 is decreased due to low activity of PI3K-m TOR signaling pathway, because of the low level of estrogen after menopause. The decreased expression of STIM1 affects the differentiation and bone formation. Our study preliminarily clarified the underlying mechanism of the role of STIM1 on osteogenesis and bone formation, futher studies are needed to clarify potential mechanisms and provide new strategy for treatment.