Faculty Bibliography

Endochondral bone formation requires the action of cells of the chondrocytic and osteoblastic lineage, which undergo continuous differentiation during this process. To identify subpopulations of resting, proliferating, and hypertrophic chondrocytes and osteoblasts involved in bone formation, we have identified here two novel marker genes present in endochondral and intramembranous ossification. Using Northern blot analysis and in situ hybridization on parallel sections of murine embryos and bones of newborn mice we compared the expression pattern of the recently cloned Itm2a and MMP-13 (collagenase-3) genes with that of established marker genes for bone formation, such as alkaline phosphatase (ALP), osteocalcin (OC), and collagen type X, during endochondral and intramembranous ossification. During embryonic development expression of Itm2a and ALP was detectable at midgestation (11.5 days postcoitum [dpc]) and increased up to 16.5 dpc. MMP-13 and OC expression started at 14.5 dpc and 16.5 dpc, respectively. This temporal expression was reflected in the spatial distribution of these markers in the growth plate of long bones. In areas undergoing endochondral ossification Itm2a expression was found in chondrocytes of the resting and the proliferating zones. Expression of ALP and MMP-13 are mutually exclusive: ALP transcripts were found only in collagen type X positive hypertrophic chondrocytes of the upper zone. MMP-13 expression was restricted to chondrocytes of the lower zone of hypertrophic cartilage also expressing collagen type X. In osteoblasts involved in endochondral and intramembranous ossification Itm2a was not present. ALP, MMP-13, and OC were mutually exclusively expressed in these cells suggesting a differentiation-dependent sequential expression of ALP, MMP-13, and OC. The identification of the continuum of sequential expression of Itm2a, ALP, MMP-13, and OC will now allow us to establish a series of marker genes that are highly suitable to characterize bone cells during chondrocytic and osteoblastic differentiation in vivo

Collagenase-3 expression in osteoblastic (UMR 106-01, ROS 17/2.8) and non-osteoblastic cell lines (BC1, NIH3T3) was examined. We observed that parathyroid hormone (PTH) induces collagenase-3 expression only in UMR cells but not in BC1 (which express collagenase-3 constitutively) or ROS and NIH3T3 cells. Since we know from UMR cells that the AP-1 factors and Cbfa1 are required for collagenase-3 expression, we analyzed the expression and PTH regulation of these factors by gel shift and Northern blot analysis in all cell lines. Gel mobility shift with a [(32)P]-labeled collagenase-3 AP-1 site probe indicated the induction of c-Fos in osteoblastic cells upon PTH treatment. While c-fos was induced in UMR cells, both c-fos and jun B were induced in ROS cells. Since Jun B is inhibitory of Fos and Jun in the regulation of the rat collagenase-3 gene in UMR cells, it is likely that high levels of Jun B prevent PTH stimulation of collagenase-3 in ROS cells. When we carried out gel shift analysis with a [(32)P]-labeled collagenase-3 RD (runt domain) site probe and Northern blot analysis with a Cbfa1 specific probe, we have observed the presence of Cbfa1 in both osteoblastic and non-osteoblastic cell lines, but there was no change in the levels of Cbfa1 RNA or protein in these cells under either control conditions or PTH treatment. From our studies above, it is evident that the expression of collagenase-3 and its regulation by PTH in osteoblastic and non-osteoblastic cells may be influenced by differential temporal stimulation of the AP-1 family members, especially c-Fos and Jun B along with the potential for posttranslational modification(s) of Cbfa1

Since estrogen is important in preventing osteoporosis in postmenopausal women and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is an estrogen antagonist in reproductive tissues, we investigated the effects of 17beta-estradiol (E2) and TCDD on collagenase-3 secretion using parathyroid hormone (PTH)-stimulated UMR 106-01 cells, a rat osteoblastic osteosarcoma cell line. Whereas E2 or TCDD had no effect on UMR cells in the absence of PTH, cells grown in the presence of 10(-7) M PTH, which induces a dramatic 30-fold increase in collagenase-3 secretion, surprisingly demonstrated a further stimulation of collagenase-3 secretion in the presence of TCDD or E2. However, the potentiating response was biphasic; i.e., at higher concentrations of E2 or TCDD, there was no enhancement of the PTH effect. PTH induces multiple effects on UMR cells, including inducing collagenase-3 mRNA transcription and regulating its extracellular abundance through a specific receptor and endocytosis. Thus, we investigated the ability of TCDD or E2 to stimulate the induction of collagenase-3 mRNA using Northern analysis. As previously reported, PTH dose dependently induced collagenase-3 mRNA after 4 h of treatment. There was little effect of TCDD or E2 on PTH-induced levels of collagenase-3 mRNA. These data could not account for the final effects on secreted collagenase-3. We postulated that low concentrations of E2 and TCDD may downregulate the collagenase-3 endocytotic two-step receptor-mediated process that includes the LDL-receptor-related protein to enhance the effects of PTH. However, this was not the case. Therefore, we conclude that low concentrations of TCDD and estrogen alter translation or secretion of PTH-stimulated collagenase-3

PTH induces c-fos expression rapidly and transiently in osteoblastic cells and requires the activity of the cAMP response element-binding protein (CREB). Here we provide evidence that protein kinase A (PKA) is the enzyme responsible for phosphorylating CREB at serine 133 (S133) and that this event is required for PTH-induced c-fos expression. PTH increases the level of phosphorylation of CREB at S133 in a time- and dose-dependent manner, correlating with the time and level of activation of PKA in response to PTH. PTH-(1-34) and -(1-31), each known to activate the cAMP pathway, induced the phosphorylation of CREB and increased the levels of c-fos messenger RNA, whereas PTH-(3-34), -(13-34), and -(28-48) could not. Specific inhibitors of calcium/calmodulin-dependent protein kinases and protein kinase C could not inhibit CREB phosphorylation or c-fos expression in response to PTH; however, H-89, a specific inhibitor of PKA, could do so in a dose-dependent manner. In addition, PTH-induced c-fos promoter activity was completely inhibited in a dose-dependent fashion by transfection of the heat-stable inhibitor of PKA. Taken together, these data provide strong evidence that PKA is the enzyme responsible for phosphorylating CREB at S133 in response to PTH and that PKA activity is required for PTH-induced c-fos expression

We have previously identified a specific receptor for collagenase-3 that mediates the binding, internalization, and degradation of this ligand in UMR 106-01 rat osteoblastic osteosarcoma cells. In the present study, we show that collagenase-3 binding is calcium-dependent and occurs in a variety of cell types, including osteoblastic and fibroblastic cells. We also present evidence supporting a two-step mechanism of collagenase-3 binding and internalization involving both a specific collagenase-3 receptor and the low density lipoprotein receptor-related protein. Ligand blot analysis shows that (125)I-collagenase-3 binds specifically to two proteins ( approximately 170 kDa and approximately 600 kDa) present in UMR 106-01 cells. Western blotting identified the 600-kDa protein as the low density lipoprotein receptor-related protein. Our data suggest that the 170-kDa protein is a specific collagenase-3 receptor. Low density lipoprotein receptor-related protein-null mouse embryo fibroblasts bind but fail to internalize collagenase-3, whereas UMR 106-01 and wild-type mouse embryo fibroblasts bind and internalize collagenase-3. Internalization, but not binding, is inhibited by the 39-kDa receptor-associated protein. We conclude that the internalization of collagenase-3 requires the participation of the low density lipoprotein receptor-related protein and propose a model in which the cell surface interaction of this ligand requires a sequential contribution from two receptors, with the collagenase-3 receptor acting as a high affinity primary binding site and the low density lipoprotein receptor-related protein mediating internalization

We investigated the regulation of collagenase-3 expression in normal, differentiating rat osteoblasts. Fetal rat calvarial cell cultures showed an increase in alkaline phosphatase activity reaching maximal levels between 7-14 days post-confluence, then declining with the onset of mineralization. Collagenase-3 mRNA was just detectable after proliferation ceased at day 7, increased up to day 21, and declined at later ages. Postconfluent cells maintained in non-mineralizing medium expressed collagenase-3 but did not show the developmental increase exhibited by cells switched to mineralization medium. Cells maintained in non-mineralizing medium continued to proliferate; cells in mineralization medium ceased proliferation. In addition, collagenase-3 mRNA was not detected in subcultured cells allowed to remineralize. These results suggest that enhanced accumulation of collagenase-3 mRNA is triggered by cessation of proliferation or acquisition of a mineralized extracellular matrix and that other factors may also be required. After initiation of basal expression, parathyroid hormone (PTH) caused a dose-dependent increase in collagenase-3 mRNA. Both the cyclic adenosine monophosphate (cAMP) analogue, 8-bromo-cAMP (8-Br-cAMP), and the protein kinase C (PKC) activator, phorbol myristate acetate, increased collagenase-3 expression, while the calcium ionophore, ionomycin, did not, suggesting that PTH was acting through the protein kinase A (PKA) and PKC pathways. Inhibition of protein synthesis with cycloheximide caused an increase in basal collagenase-3 expression but blocked the effect of PTH, suggesting that an inhibitory factor prevents basal expression while an inductive factor is involved with PTH action. In summary, collagenase-3 is expressed in mineralized osteoblasts and cessation of proliferation and initiation of mineralization are triggers for collagenase-3 expression. PTH also stimulates expression of the enzyme through both PKA and PKC pathways in the mineralizing osteoblast

c-fos is induced in response to parathyroid horinonr (PTH) in a (AMP-depetident manner in the rat osteosarroina cell line UMR 106-01. PTH treatment leads to the phosphorylation of the cAMP response element binding prutrinf(CREB) at M-rine 133 (pCRKB) which binds the major CHK of c-fos leading to increased trrtn.srription. Here we provide evidence that phosphoryiatjon of CRKH by cAMP-dependent protein kinase (PKA) is required for the induction of c-fus expression by PTH. In UM R, 106-01 cells the PKC activator phorbol mylistate acetate and the cAMP analog 8-bromo-rAMP bol h induced the phosphorylation of CRKH while iononiycin did not, suggesting CREH is pbosphoryhited by PKA or PKC. 11-89. a specific inhibitor of PKA. inhibited pCRKU iormation and r-fos expression, whereas specific inhibitors of ralciuni/calniodulin dependent prolein kinases and PKC could not, Vsing various fragments of PTll wliich have been shown to activate different second messenger pathways. only PTH fragnieuls which can increa.se cAMP levels could increase both c-fos mRNA level and pCREB. Further, a c-fos promoter reporter plasrnid was cotransfected with increasing amounts of the human heat-.stable inhibitor of protein kinase A (PK'I). PTH-induced promoter activity was completely inhibited in a dose-dependent manner by PK1. In addition. GAL4-CREB chimeras were employed to eliminate the effects of endogenous CRKH. GAL4-CREB can confer PIH responsiveness to a he.terologous promoter, while a S133A mutant cannot. Taken together, these data provide strong evidence that PTH requires the activity of PKA to phosphoryiate CRKB at S 133 and increase levels of c-fos mKNA.

Exposure to zero gravity has been shown to cause a decrease in bone formation. This implicates osteoblasts as the gravity-sensing cell in bone. Osteoblasts also are known to produce neutral proteinases, including collagenase and tissue plasminogen activator (tPA), which are thought to be important in bone development and remodeling. The present study investigated the effects of zero gravity on development of calvariae and their expression of collagenase and tPA. After in utero exposure to zero gravity for 9 days on the NASA STS-70 space shuttle mission, the calvariae of rat pups were examined by immunohistochemistry for the presence and location of these two proteinases. The ages of the pups were from gestational day 20 (G20) to postnatal (PN) day 35. Both collagenase and tPA were found to be present at all ages examined, with the greatest amount of both proteinases present in the PN14 rats. At later ages, high amounts were maintained for tPA but collagenase decreased substantially between ages PN21 to PN35. The location of collagenase was found to be associated with bone-lining cells, osteoblasts, osteocytes, and in the matrix along cement lines. In contrast, tPA was associated with endothelial cells lining the blood vessels entering bone. The presence and developmental expression of these two proteinases appeared to be unaffected by the exposure to zero gravity. The calvarial thickness of the pups was also examined; again the exposure to zero gravity showed little to no effect on the growth of the calvariae. Notably, from G20 to PN14, calvarial thickness increased dramatically, reaching a plateau after this age. It was apparent that elevated collagenase expression correlated with rapid bone growth in the period from G20 to PN14. To conclude, collagenase and tPA are present during the development of rat calvariae. Despite being produced by the same cell in vitro, i.e., the osteoblast, they are located in distinctly different places in bone in vivo. Their presence, developmental expression, and quantity do not seem to be affected by a brief exposure to zero gravity in utero