Progress 10/01/09 to 09/30/12
Outputs
OUTPUTS: This research has provided training to several undergraduate and graduate students in research. Specifically, four graduate students completed their master's thesis research which related to the objectives of this project. Additionally, eight undergraduate students have received training in laboratory techniques, animal handling, data collection, data analysis and presentation of research data at regional and national meetings. Four of the undergraduates received UConn Office of Undergraduate Research Grants for their research related to this project. One undergraduate completed an honors thesis and presented the findings at the University of CT Frontiers in Undergraduate Research Poster Presentation. Results have been presented at the national ASAS meetings, University seminars and classroom lectures. PARTICIPANTS: Individuals: Principle Investigator - Kristen Govoni. Partner Organizations: University of Connecticut Health Center provided microCT analysis of poultry bones. Collaborators: Subburaman Mohan, Jerry L Pettis VA Medical Center; Collaborators: Michael Darre, Steven Zinn, Thomas Hoagland, Kumar Venkitanarayan, Gary Kazmer, Univ. of CT. Training: Elizabeth Ackell, Maria Procopio, Nidhish Francis, Sarita Neupane - obtained a master's degree. Chelsea Mora, Dana Kaelin, Guiuliana Miranda, Stephanie Tournaquindici, Stephanie Spignesi, and Katelyn McFadden - undergraduate students received training in laboratory techniques, data collection and analysis. Amanda Lopez - undergraduate honors students completed her honors thesis. Amanda Lopez, Stephanie Tournaquindici, Stephanie Spignesi, and Katelyn McFadden presented data at UConn Frontiers Poster presentation. Andrew Galinsky - high school student gained experience in research, data analysis and presentation. TARGET AUDIENCES: Target audiences include faculty at the University of Connecticut and other institutions, scientists, graduate students, and undergraduates. Data generated from these experiments has been presented at national scientific meetings, in peer-reviewed journals, in departmental seminars at UConn and other peer institutions. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We used several models to further our understanding of bone development and maintenance. Using broilers, we hypothesized that oral supplementation of LiCl would increase bone strength and quality in broiler chickens. 144 broilers were divided into LiCl, control (C) and pair-fed (PF) groups. Beginning at 1 or 3 weeks (wk) of age, chickens were administered LiCl (20 mg/kg BW) or water daily by oral gavage. At 6 wk of age, chickens were euthanized and blood, bone and muscle samples were collected. We did not observe any effect of LiCl treatment on BW (p ≥ 0.53), feed intake (p > 0.19) or muscle color or lipid oxidation (p > 0.05), demonstrating that LiCl treatment did not negatively affect growth in these broilers. Using microCT imaging, we did not observe a difference in cortical or trabecular bone volume, trabecular thickness, number, or spacing (p > 0.52). Using 3-point bending, we did not measure a difference in bone length or ultimate load (p > 0.60). However, we did observe a 23% reduction in stiffness (p = 0.02) in the femora and 34% reduction in fracture energy (p = 0.11) in the tibiae of the LiCl treated birds, thus suggesting reduced bone quality in the LiCl birds. In conclusion, LiCl treatment reduced bone stiffness, which may be due to the dose of LiCl utilized or a species difference in response to LiCl treatment on bone formation. In horses, we successfully isolated and cultured bone marrow mesenchymal stem cells from horses and were able to differentiate these cells into osteoblasts in culture. We determined that dexamethasone (P < 0.001), but not hBMP-2 (P > 0.05) increased ALP. In addition, during differentiation, expression of Tbx3, a gene involved in regulating osteoblast function was 4-fold less at d 18 (P < 0.01). In summary, dexamethasone is essential for differentiation into osteoblast cells, and inhibition of Tbx3 may be required for optimal differentiation of eBMSC. Using a bovine model, we further evaluated the role of Tbx2 and Tbx3, two key transcription factors in bone and mammary gland development. We hypothesized that GH and IGF-I would increase Tbx2 and Tbx3 expression in bovine MEC. MAC-T cells were treated with GH at 100 (GH100) or 500 (GH500) ng/mL or IGF-I at 100 (IGF100) or 200 (IGF200) ng/mL for 24 and 48 hours. As determined by real-time RT-PCR, we did not observe a change in Tbx3 expression in cells treated with GH (P ≥ 0.74). However, both IGF-I treatments increased Tbx3 expression (P ≤ 0.03). Surprisingly, expression of Tbx2 was not detectable in primary MEC cells; however it was expressed in mammary fibroblast cells. In fibroblast cells treated with GH500 and IGF200, we did observe a change in Tbx2 or Tbx3 expression (P ≥ 0.76). In conclusion, IGF-I regulates Tbx3 expression in bovine MEC and Tbx2 and Tbx3 expression are cell type specific. Lastly, using Saos2 cells, knockdown of Tbx2 expression and protein using siRNA reduced cell proliferation, thus demonstrating that Tbx2 is required for cell proliferation of Saos2 cells. Overall, we determined that Tbx proteins play a key role in bone cell function in several species.
Publications
- Francis, N, Tornaquindici, SM, Mohan, S, and Govoni, KE. 2012. T-box (Tbx)2 is required for proliferation of osteoblast cells. J. Anim. Sci. Vol. 90, Suppl. 3.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: This research has provided training to several undergraduate and graduate students in research. Specifically, two graduate students completed their master's thesis research which related to the objectives of this project. Currently one student is working on a master's degree and one student is working on a Ph.D. Additional, six undergraduate students have received training in laboratory techniques, animal handling, data collection, data analysis and presentation of research data at regional and national meetings. Four of the undergraduates received UConn Office of Undergraduate Research Grants for their research related to this project. One undergraduate completed an honors thesis and presented the findings at the University of CT Frontiers in Undergraduate Research Poster Presentation. Results have been presented at the national ASAS meeting, University seminars and classroom lectures. PARTICIPANTS: Individuals: Principle Investigator - Kristen Govoni. Collaborators: Subburaman Mohan, Jerry L Pettis VA Medical Center; Steven Zinn, Thomas Hoagland, Kumar Venkitanarayan, Gary Kazmer, Univ. of CT. Training: Elizabeth Ackell and Maria Procopio - obtained a master's degree. Sarita Neupane - working on master's thesis research. Maria Procopio - working on dissertation research. Dana Kaelin, Guiuliana Miranda, Stephanie Tournaquindici, Stephanie Spignesi, and Katelyn McFadden - undergraduate students received training in laboratory techniques, data collection and analysis. Amanda Lopez - undergraduate honors students completed her honors thesis and presented data at UConn Frontiers Poster presentation. Andrew Galinsky - high school student gaining experience in research, data analysis and presentation. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Significant findings using equine model: In continuation of our previous work to determine the ideal conditions to culture and differentiation equine bone marrow stromal cells into osteoblasts, we evaluated the effects of dexamethasone and bone morphogenic protein (BMP)-2 on alkaline phosphatase activity (ALP). Treatment with dexamethasone (P < 0.001), but not hBMP-2 (P > 0.05) increased ALP activity compared with control cells. In addition, during differentiation, expression of runx2 increased 3-fold (P < 0.001) and osteocalcin, a marker of bone formation, increased 260-fold by d 18 of culture (P < 0.001) demonstrating successful differentiation into osteoblasts in our culture system. In addition, expression of Tbx3, a gene involved in regulating osteoblast function, increased 1.8-fold at d 3 (P < 0.01), however expression was 4-fold less at d 18 (P < 0.01). In summary, dexamethasone is essential for differentiation into osteoblast cells, and inhibition of Tbx3 may be required for optimal differentiation of eBMSC. Significant findings using bovine model: It is known that Tbx2 and 3 are expressed in mammary gland, therefore we wanted to determine if these genes were involved in regulating key growth factors [growth hormone (GH) and insulin-like growth factor-I (IGF-I)] in mammary epithelial cells (MEC). We hypothesized that GH and IGF-I would increase Tbx2 and Tbx3 expression in bovine MEC, the cell responsible for milk production. To test our hypothesis, MAC-T cells (MEC line) were treated with GH at 100 (GH100) or 500 (GH500) ng/mL or IGF-I at 100 (IGF100) or 200 (IGF200) ng/mL for 24 and 48 hours. As determined by real-time RT-PCR, we did not observe a change in Tbx3 expression in cells treated with GH (P > 0.74). However, both IGF-I treatments increased Tbx3 expression (P < 0.03). Surprisingly, expression of Tbx2 was not detectable in primary MEC cells; however it was expressed in mammary fibroblast cells. In fibroblast cells treated with GH500 and IGF200, we did observe a change in Tbx2 or Tbx3 expression (P > 0.76). In conclusion, IGF-I regulates Tbx3 expression in bovine MEC and Tbx2 and Tbx3 expression are cell type specific. Significant findings using human cell model: Based on our previous findings that we were not able to detect Tbx2 protein in mouse osteoblasts cells (MC3T3-E1), we evaluated Tbx2 in the human osteosarcoma cell line, Saos2 cells, and we were able to detect mRNA expression and protein by western blot. Further studies determined that knockdown of Tbx2 expression and protein using siRNA, reduced cell proliferation, thus demonstrating that Tbx2 is required for cell proliferation of the Saos2 cells.
Publications
- 1) Govoni, KE. 2011. Insulin-like growth factor-I molecular pathways in osteoblasts: Potential targets for pharmacological manipulation. Curr. Mol. Pharmacol. Jun 25 [Epub ahead of print].
- 2) Govoni, KE, Goodman, D, Maclure RM, Penfold, LM, and Zinn, SA. 2011. Serum concentrations of insulin-like growth factor and insulin-like growth factor binding protein-2 and -3 in eight hoofstock species. Zoo Biol.. 30:275-84.
- 3) Ackell, ER, Sanchez, A, Mora, C, Zinn, SA, Hoagland, T, and Govoni, KE. 2011. Expression of key transcription factors during differentiation of equine bone marrow mesenchymal stem cells (eBMSC) into osteoblast cells. J. Anim. Sci., Vol 89, E-Supp 1:717.
- 4) Procopio, ML, Lopez, AC, McFadden, K, Kazmer, GW, Hoagland, TA, and Govoni, KE. 2011. Expression of T-box3 (Tbx3) in bovine mammary epithelial cells. J. Anim. Sci., Vol 89, E-Supp 1:208.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: This research has provided training to several undergraduate and graduate students in research. Four undergraduate students have been trained in laboratory techniques, animal handling, data collection, data analysis and presentation of research data. One undergraduate received a summer research grant through the University of CT for her work with poultry. Her work was presented at a University of CT Frontiers in Undergraduate Research Poster Presentation and at the ASAS National Meeting in Denver 2010. Abstract: "Effect of daily lithium chloride (LiCl) administration on bone quality and strength in growing broiler chickens". One graduate student completed his master's degree titled "Effect of Tbx2 in osteoblast function". Two graduate students are working on their master's thesis research related to the objectives of this project. Results have been presented at the national ASAS meeting, University seminars and classroom lectures. PARTICIPANTS: Individuals: Principle Investigator - Kristen Govoni. Partner Organizations: University of Connecticut Health Center provided microCT analysis of poultry bones. Collaborators: Subburaman Mohan, Jerry L Pettis VA Medical Center; Michael Darre, Steven Zinn, Thomas Hoagland, Kumar Venkitanarayan, Gary Kazmer, Univ. of CT. Training: Nidhish Francis - obtained a master's degree. Elizabeth Ackell - working on master's thesis research. Maria Procopio - working on master's thesis research. Beth Harvey - undergraduate student gained experience in research, data collection and presentation. She received an undergraduate research grant for her work and presented data at a national meeting. Chelsea Mora, Guiuliana Miranda, Stephanie Tournaquindici - undergraduate students received training in laboratory techniques, data collection and analysis. Amanda Lopez - undergraduate honors students received training and collected data for her honors thesis project. Katelyn McFadden - high school student gained experience in research, data analysis and presentation. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Significant findings using poultry model: Bone fractures and deformities are a serious problem for the broiler industry; therefore, identification of mechanisms to improve bone quality and strength would be beneficial. The wnt/beta-catenin pathway plays a critical role in the bone formation process and this pathway can be stimulated by oral LiCl supplementation in mice. We hypothesized that oral supplementation of LiCl would increase bone strength and quality in broiler chickens. 144 broilers were divided into LiCl, control (C) and pair-fed (PF) groups. Beginning at 1 or 3 weeks (wk) of age, chickens were administered LiCl (20 mg/kg BW) or water daily by oral gavage. At 6 wk of age, chickens were euthanized and blood, bone and muscle samples were collected. A 24h LiCl (20 mg/kg BW) challenge determined that serum LiCl increased within 2h and cleared the system within 24h, thus demonstrating the effectiveness of our oral gavage to deliver LiCl. We did not observe any differences in BW (p ≥ 0.53) or feed intake (p > 0.19) between all treatment groups, demonstrating that LiCl treatment did not negatively affect growth in these broilers. To evaluate bone composition, we performed morphometric analysis on the tibiae of C and LiCl groups using microCT imaging. We did not observe a difference in cortical or trabecular bone volume, trabecular thickness, number, or spacing (p > 0.52). To determine bone strength, we performed 3-point bending on the femora and tibiae of C and LiCl birds from the 1 wk group. We did not measure a difference in bone length or ultimate load (p > 0.60). However, we did observe a 23% reduction in stiffness (p = 0.02) in the femora and 34% reduction in fracture energy (p = 0.11) in the tibiae of the LiCl treated birds, thus suggesting reduced bone quality in the LiCl birds. We did not observe any effect of LiCl treatment on pectoralis muscle color or lipid oxidation (p > 0.05). In conclusion, LiCl treatment in broilers did not affect growth or meat quality. Surprisingly, we measured a reduction in bone stiffness with LiCl treatment, which may be due to the dose of LiCl utilized or a species difference in response to LiCl treatment on bone formation. Significant findings using horse model: We successfully isolated and cultured bone marrow stormal cells (BMSC) from horses. The proliferation potential of these cells is greater with purchased calf serum compared with in house horse serum (P > 0.05). Equine BMSC can be induced to differentiate into osteoblasts in the presence of ascorbic acid and beta-glycerol phosphate. The expression of key transcription factors involved in differentiation to osteoblasts is currently being examined. Significant findings using murine and human models: The protein expression of Tbx2, a key transcription factor in embryonic development, is detectable in mouse lung and Saos-2 (human osteosarcoma) cells, but not detectable in mouse bone, BMSC and MC3T3-E1 (mouse osteoblast) cells. Further studies are underway to determine the effect of knockdown of Tbx2 on cell function using Saos-2 cells.
Publications
- No publications reported this period
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