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Decreased estrogen levels in post-menopausal women are associated with an imbalance of bone turnover with the potential to develop into osteoporosis. Increased osteoclastogenesis leads to increased bone resorption and consequential loss of bone density. A key regulator of osteoclast recruitment is the osteoclast differentiation factor (ODF). This lab previously reported that mechanical strain inhibits ODF expression through activation of extracellular signal regulated protein kinases (ERK1/2) in bone marrow stromal cells. The ERK1/2 inhibitor PD98059 (ERKI) was used to show that inactivation of MAPK decreases the strain effect on ODF mRNA. In osteoblastic cells, estrogen has been found to activate MAPK by phosphorylation. Because the time course and concentration-response occurred at a smaller range than that required for transcriptional activation, this activation may be mediated through a cell membrane receptor rather than an ER. In this report, we explored the effects of estradiol with ERKI on ODF expression. ST2 stromal cells support osteoclast formation when grown under the influence of osteotropic factors, which up-regulate the ODF gene. These cells were cultured and treated with 1,25 dihydroxyvitamin D, estrogen, and/or ERKI. Northern analysis was performed to measure effects on mRNA. Western blots were performed to assess estrogenís modulation of MAPK. We present preliminary evidence that estrogen amplifies ERKI stimulation of ODF mRNA expression. Estrogen also stimulated ERK protein expression. The next step is to determine if MAPK has similar effects to estrogen. ST2 cells will be infected with an adenovirus vector to transfer dominant-negative and constitutive MAPK promoters. These infected cells will then be treated with estrogen to discover if inhibition of MAPK prevents estrogenís effects.
The presence of both N2 and HI 41 SNP versions in the Unc non-Spe’s and the presence of only HI SNP versions in the Dpy non-Spe’s shows that spe-5 could be to the right of the 41 SNP. More concrete data is needed, however, before spe-5’s position relative to the 41 SNP can be considered conclusive. Such data is forthcoming from additional non-Spe and Spe strains waiting to be typed. The discovery of an Unc Spe Mat recombinant in the mat-1 mapping process is encouraging, but not conclusive, in that spe-5 could be to the left of mat-1. Therefore, spe-5 is now hypothesized to be in an interval between the 41 SNP and mat-1.
Andy Golden at the National Institutes of Health provided us with mat-1 for mapping against spe-5. Funding to support the student was provided by Emory University’s Hughes Sciences Initiative SURE 2000 Program. I thank the directors of the program for their efforts to open more avenues for undergraduate students to gain research experience. The lab in which this project was conducted is supported by a grant from the National Institutes of Health.
Julia investigated how estrogen and ERK1//2 regulate the expression of osteoclast differentiation factor (ODF). She used ST2 cells, which are cloned cell line of bone stromal cells. These cells were treated with vitamin D, which stimulate ODF expression, plus estrogen, ERKi (ERK inhibitor), or both. She used Northern analyis to see how these treatments affected ODF mRNA treatment. Compared to control values, the estrogen decreased ODF and ERKi increased ODF as she hypothesized. Combined, ERKi block estrogen's effects. She then wanted to see how estrogen affected inactive ERK1/2. In order to do this, she used adenovirus labeled with green fluorescent protein (Ad-GFP) to infect ST2 cells. She used flow cytometry analysis to determine how much adenovirus was necessary to infect the cells. Her lab is infecting the cells with dominant negative ERK Ad-GFP to see the effects on ODF expression. Mapping spe-5 against mat-1. mat-1 is known to reside in a cosmid gap in LG I between unc-38 and dpy-5 . At 25°C, the recessive, temperature-sensitive allele mat-1(ax212) causes either unfertilized oocytes due to spermatogenesis defects or lethality at the one-cell embryo stage. mat-1(ax212) has no abnormal phenotypes at 16°C. spe-5 was mapped against mat-1(ax212).
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