Progress 10/01/02 to 09/30/07
Outputs
Studies were conducted to examine the effect of prolactin (1-1000 ng/mL) cultured in either fetal bovine serum or horse serum, on expression of key genes in development of the osteoblast phenotype (i.e., alkaline phosphatase, bone sialoprotein, and osteocalcin) and development of mineralized nodules in vitro, using primary cultures of osteoblast-like cells isolated from fetal rat calvaria. Experimental conditions were modified (such as altering serum type during proliferation versus differentiation, timing of prolactin dose during cell proliferation versus differentiation, and using serum-free conditions during periods of early differentiation). Results indicated that osteoblast differentiation was significantly influenced by serum type, although any additional prolactin effect, beyond that of serum source, on development of the osteoblast phenotype was negligible. Additional studies were conducted to examine the impact of prolactin on altered bone formation in the
presence of inflammation. Basal mRNA expression of IL-1a, IL-1b, and TGF-B were evident late in culture of osteoblast-like cells derived from bone marrow of 2 yr old male rats. This gene expression was not detected in similar cultures from younger male rats. Basal mRNA levels for these cytokines were either not expressed or expressed in very low levels in cultures of osteoblasts from fetal rat calvaria. Results suggest the bone marrow microenvironment of the older rat may have more constitutive cytokine expression (or a great capacity to stimulate cytokine production) than that of younger rats. In subsequent studies, a nonspecific inflammatory response was induced by treatment with lipopolysaccharide (LPS) in osteoblast-like cells isolated from fetal rat calvaria. Results indicate that LPS alone (without co-treatment with a synthetic glucocorticoid, dexamethasone) significantly inhibited osteoblast development and function in vitro. This was evident by 1) lower than normal expression
of markers of osteoblast phenotypic development, including osteocalcin (osteocalcin is a specific, late marker that is up-regulated during osteoblast maturation), and 2) suppressed osteoblast function, as assessed by formation of fewer and smaller multilayered, mineralized nodules in vitro. In the presence of dexamethasone, response to LPS stimulation was blunted. When cells were co-treated with LPS and dexamethasone, osteocalcin gene expression and mineralized nodule development were similar to controls; prolactin produced no further effect. Collectively, results of these studies suggest that prolactin, per se, may not directly impact osteoblast phenotypic development or function when studied in the cell system used (primary cultures of osteoblast-like cells isolated from fetal rat calvaria). Whether prolactin impacts the skeleton of aged rats or with conditions of inflammation is unknown, but warrants further investigation. An alternative hypothesis for this research is that
prolactin enhances the production and secretion of bone-resorptive cytokines, such as interleukin 1, transforming growth factor-beta, RANK ligand, and osteoprotegrin, which would enhance osteoclastic bone resorption in vivo.
Impacts
Because circulating prolactin levels increase with age, and excessively high prolactin levels are known to cause bone loss, it is important to understand the impact of age-related increases in prolactin on age-related bone loss. Although the current research has not fully ascertained the impact of prolactin on the aging bone, it has provided evidence that prolactin may not have a major impact on bone when studied using primary cultures of osteoblast-like cells isolated from fetal rat calvaria. Determination of the mechanism of action of prolactin in the aged bone or with conditions of inflammation may provide insights into new therapeutic approaches to prevent or minimize age-related bone loss and osteoporosis and ultimately decreasing morbidity and mortality from complications of osteoporosis and related medical costs.
Publications
- OAS JL, 2007, The effects of serum, prolactin, and troglitazone on phenotype development in osteoblast-like cells derived from fetal rat calvaria (M.S. Thesis)
|
Progress 10/01/05 to 09/30/06
Outputs
Bone formation decreases in older adults, with a concomitant increase in fatty bone marrow. Prolactin, a hormone that increases in human beings with age, appears to promote the development of age-related bone loss and the development of fatty bone marrow. Additionally, recent studies identify that inflammatory processes, due to increased cytokine production in older bone microenvironments, may also contribute to hormonally-induced age-related bone loss. The primary focus of studies conducted during this reporting period has been to examine the effect of inflammation on osteoblast development and function as well as expression of genes for key bone-resorptive cytokines, using primary cultures of osteoblast-like cells isolated from fetal rat calvariae. A nonspecific inflammatory response was induced by treatment with lipopolysaccharide (LPS). Results indicate that LPS alone (without co-treatment with a synthetic glucocorticoid, dexamethasone) significantly inhibits
osteoblast development and function in vitro. This is evident by 1) lower than normal expression of markers of osteoblast phenotypic development, including osteocalcin (osteocalcin is a specific, late marker that is upregulated during osteoblast maturation), and 2) suppressed osteoblast function, as assessed by formation of fewer and smaller multilayered, mineralized nodules in vitro. In the presence of dexamethasone, response to LPS stimulation is blunted. Results indicate that osteocalcin gene expression was either only slightly lower or the same as control cells and mineralized nodule development was similar to normal, when cells were co-treated with LPS and dexamethasone. In addition to evaluating osteoblast phenotypic development, the effect of LPS, with and without dexamethasone, on key inflammatory hormones (IL-1a, IL-1b, and TGF-B) is currently underway. Results thusfar suggest that inflammation in the bone marrow microenvironment may result in compromised osteoblast
development and function. This raises the possibility that inflammation may contribute to age-related bone loss through an interaction with an altered bone microenvironment, which may include higher levels of cytokines with aging. Since an elevation in prolactin occurs with aging, future studies will be conducted to examine the interaction of glucocorticoids and prolactin treatment, in LPS-stimulated cells, on maturation and function of the differentiating osteoblast. These studies are important in understanding the extent that changes in the hormonal profile of the bone microenvironment with aging (such as increased prolactin, glucocorticoids, and overall inflammatory response) influence age-related bone loss.
Impacts
Knowledge of the extent to which high prolactin levels and it's interaction with age-related inflammatory processes, contribute to age-related bone loss, and potentially with the development of osteoporosis, will provide insights into new therapeutic approaches to prevent or minimize age-related bone loss and osteoporosis. Ultimately, this has the potential to substantially decrease morbidity and mortality from complications of osteoporosis and related medical costs.
Publications
- No publications reported this period
|
Progress 10/01/04 to 09/30/05
Outputs
Bone formation decreases in older adults, with a concomitant increase in fatty bone marrow. Prolactin, a hormone that increases in human beings with age, appears to promote the development of age-related bone loss and the development of fatty bone marrow. However, the mechanism through which prolactin mediates its osteopenic effect in older animals is not known. In general, prolactin may work through altering osteoblast maturation and function, thus altering matrix formation and bone mineralization. Alternatively, prolactin may increase osteoblastic production of bone-resorptive cytokines; these cytokines enhance osteoclastic bone resorption in vivo. The primary focus of studies conducted during this reporting period has been to examine the basal and lipopolysaccharide (LPS)-stimulated expression of genes for key bone-resorptive cytokines, such as interleukin-1 alpha (IL-1a), interleukin-1 beta (IL-1b), and transforming growth factor-beta (TGF-B). Results indicate
that basal mRNA levels for these cytokines are either not expressed or expressed in very low levels in cultures of osteoblasts from fetal rat calvaria. In contrast, basal mRNA expression of IL-1a, IL-1b, and TGF-B was evident late in culture of osteoblast-like cells derived from bone marrow of 2 yr old male rats; this expression was not detected in similar cultures from younger male rats. These results suggest the bone marrow microenvironment of the older rat may have more constitutive cytokine expression (or a great capacity to stimulate cytokine production) than that of younger rats. This raises the possibility that prolactin may contribute to age-related bone loss through an interaction with an altered bone microenvironment, which may include higher basal level of cytokines with aging. In order to further explore this, studies are underway using LPS to stimulate cytokine expression in osteoblasts from fetal rat calvaria. This stimulation would then allow for studies to examine the
interaction of glucocorticoids and prolactin treatment, in LPS-stimulated cells, on maturation and function of the differentiating osteoblast. Studies have thusfar identified that a dose of 0.1 ug/mL LPS results in maximal stimulation of IL-1a, IL-1b, and TGF-B in osteoblasts derived from fetal rat calvaria, and this expression is maximal at 3 hr after treatment. In the presence of dexamethasone, response to LPS stimulation is blunted. Outcomes to evaluate the effect of LPS on osteoblast maturation and function include an evaluation of the expression of key genes in development of the osteoblast phenotype (i.e., alkaline phosphatase, bone sialoprotein, and osteocalcin) and development of mineralized nodules in vitro. Studies to evaluate the interactions of prolactin and glucocorticoid treatment, using LPS-stimulated osteoblasts, on development of the osteoblast phenotype are currently underway.
Impacts
Prolactin is a hormone that increases with age, and very high serum levels of prolactin even in younger humans are associated with low bone mass. Knowledge of the extent to which high prolactin levels contribute to age-related bone loss, and potentially with the development of osteoporosis, will provide insights into new therapeutic approaches to prevent or minimize age-related bone loss and osteoporosis. Ultimately, this has the potential to substantially decrease morbidity and mortality from complications of osteoporosis and related medical costs.
Publications
- No publications reported this period
|
Progress 10/01/03 to 09/30/04
Outputs
Bone formation decreases in older adults, with a concomitant increase in fatty bone marrow. Prolactin, a hormone that increases in human beings with age, appears to promote the development of age-related bone loss and the development of fatty bone marrow. However, the mechanism through which prolactin mediates its osteopenic effect in older animals is not known. In general, prolactin may work through altering osteoblast maturation and function, thus altering matrix formation and bone mineralization. Alternatively, prolactin may alter bone through production of bone-resorptive cytokines that enhance osteoclastic bone resorption in vivo. The primary focus of studies conducted during this reporting period has been to example the extent to which prolactin impacts development of the osteoblast phenotype. Primary cultures of osteoblast-like cells isolated from fetal rat calvaria have been used in numerous studies to examine the effect of prolactin (1-1000 ng/mL) cultured in
either fetal bovine serum or horse serum, on expression of key genes in development of the osteoblast phenotype (i.e., alkaline phosphatase, bone sialoprotein, and osteocalcin) and development of mineralized nodules in vitro. Many different experimental approaches have been used (such as altering serum type during proliferation versus differentiation, altering the timing of prolactin dose during cell proliferation versus differentiation, and doing serum-free studies during periods of early differentiation). Overall, results indicate that osteoblast differentiation is significantly influenced by serum type, although any prolactin effect on development of the osteoblast phenotype is negligible. The alternative hypothesis for this research is that prolactin enhances the production and secretion of bone-resorptive cytokines, such as interleukin 1, transforming growth factor-beta, RANK ligand, and osteoprotegrin, which would enhance osteoclastic bone resorption in vivo. Initial studies are
underway to examine this hypothesis, examining prolactin-related effects on expression of these genes and production and secretion of these proteins.
Impacts
Prolactin is a hormone that increases with age, and very high serum levels of prolactin even in younger humans are associated with low bone mass. Knowledge of the extent to which high prolactin levels contribute to age-related bone loss, and potentially with the development of osteoporosis, will provide insights into new therapeutic approaches to prevent or minimize age-related bone loss and osteoporosis. Ultimately, this has the potential to substantially decrease morbidity and mortality from complications of osteoporosis and related medical costs.
Publications
- No publications reported this period
|
Progress 10/01/02 to 09/30/03
Outputs
Bone formation decreases in older adults, with a concomitant increase in fatty bone marrow. The common bipotential progenitor of osteoblasts and adipocytes in the bone marrow is a major focus of osteoporosis research. Prolactin, a hormone that increases in human beings with age, appears to promote the development of age-related bone loss and the development of fatty bone marrow. However, the mechanism through which prolactin mediates its osteopenic effect in older animals is not known. We have conducted preliminary cell culture studies and initial experiments to evaluate the effect of prolactin on osteoblastogenesis. Preliminary studies were conducted to determine the appropriate cell culture conditions under which to study prolactin-related effects. There is a substantial amount of prolactin and other lactogenic hormones in fetal bovine serum (FBS), which is used in high concentration (10%) in calvarial cell cultures. We examined the impact of using horse serum (HS),
which has much lower lactogenic hormone levels than FBS, on calvarial cultures. When HS was used throughout the culture period (entire 3 wk of culture), cells did not develop into mature osteoblasts. These cells did not express the normal pattern of genes that were up- or down-regulated during osteoblast maturation, and the cultures did not form multi-layered mineralized nodules, an end-point measurement of bone formation in vitro. When HS was used only during the proliferation period (first 8-10 days) and switched to FBS for the differentiation period (days 10-21 of culture), cells were slow to differentiate and mineralized nodule formation was about 50% of normal. When HS was used during differentiation only, cells differentiated with the normal pattern of gene expression and nodule development and mineralization. Thus, use of HS is least disruptive to osteoblast maturation when used only during differentitation. A second set of experiments tested whether prolactin suppresses normal
osteoblast differentiation and mineralized nodule formation. The concentration-dependent effect of prolactin on osteoblast differentiation and nodule formation was evaluated during proliferation only, during differentiation only, or both during proliferation and differentiation. Results indicated that treatment with prolactin (with HS) only during proliferation and then switched to FBS (without prolactin) during differentiation resulted in a 85% suppression of nodule development compared to controls. This is a much greater suppression than occurred with HS alone (46% lower nodule formation), suggestive of a much greater suppressive effect of prolactin plus HS on osteoblastogenesis than occurs with HS. Treatment with prolactin (with HS) only during differentiation did not alter gene expression or nodule formation, suggesting that the target time for prolactin-related suppression may be during early osteoblast formation. This suppression was not rescued by discontinuation of prolactin
or switching to FBS after the proliferative period. Studies are being conducted to further characterize this prolactin-related effect on early osteoblast development.
Impacts
As an outcome of the proposed studies, mechanisms by which prolactin influences age-related decreased bone formation and increased fatty bone marrow will be determined. The research proposed in this application is significant, because knowledge of the mechanism for the age-related bone changes is expected to provide insights into new therapeutic approaches to prevent or minimize age-related bone loss in the treatment of osteoporosis. Ultimately, this has the potential to substantially decrease morbidity and mortality from complications of osteoporosis and related medical costs.
Publications
- Oas, J. L., Dumais, B., Cooney, P., Aubin, J. E. and Kipp, D. E. (2003). Horse serum suppresses bone nodule formation and osteoblast differentiation in fetal rat calvarial cell cultures. FASEB J 17:A1093-A1094.
|
|