Nicola C. Partridge

The physician-scientist: career issues and challenges at the year 2000.

In the midst of a promising era of expansion in biomedical research, there is growing concern about a serious decline in a crucial category of research personnel: physician-scientists. If this trend continues, many believe that key types of medical research will suffer (1). While apprehension about the survival of physician-scientists had been expressed previously (2–4), there are a number of indications that this problem is becoming more severe. Anecdotal evidence of the difficulties in recruiting and retaining medical school faculty has been growing. Increasingly, prominent leaders in the research community are calling attention to this issue (5, 6). External forces, leading to changes in the finances of academic health centers, are raising new obstacles to research and training (7). Nominations of physicianscientists age 45 or younger to honorary societies such as the American Society for Clinical Investigation have declined by almost 30% over the past decade, suggesting that the pool of talented young investigators is shrinking. The implications of this situation for the progress of medical research made the question relevant to the mission of the Federation of American Societies for Experimental Biology (FASEB): To enhance the ability of biomedical and life scientists to improve, through their research, the health, well-being, and productivity of all people. At its December 1998 meeting, the FASEB Board voted to initiate an investigation of physicianscientists and career opportunities for biomedical research. Responsibility for this study was given to the Career Opportunities Subcommittee of the Science Policy Committee, under the leadership of Nicola Partridge. The subcommittee collected and analyzed data on training and research activities from the Association of American Medical Colleges (AAMC), the American Medical Association (AMA), and the National Institutes of Health (NIH). In addition, the subcommittee organized a conference to address the following questions: • Are physician-scientists critical to the success of the biomedical research enterprise? • What evidence exists that there is a decline in physician-scientists? • If there is a decline, how might it be reversed or alleviated? Delegates from the FASEB societies held a closed session at the conclusion of the conference in order to review the data and the panelists’ testimony and to formulate recommendations. Through ongoing communication with the delegates, the Career Opportunities Subcommittee has endeavored to present, along with the data and conclusions from the conference, the consensus-based recommendations of the FASEB society delegates.

Parathyroid Hormone Regulates the Rat Collagenase-3 Promoter in Osteoblastic Cells through the Cooperative Interaction of the Activator Protein-1 Site and the runt Domain Binding Sequence

Parathyroid hormone induces collagenase-3 gene transcription in rat osteoblastic cells. Here, we characterized the basal, parathyroid hormone regulatory regions of the rat collagenase-3 gene and the proteins involved in this regulation. The minimal parathyroid hormone-responsive region was observed to be between base pairs −38 and −148. Deleted and mutated constructs showed that the activator protein-1 and the runt domain binding sites are both required for basal expression and parathyroid hormone activation of this gene. The runt domain site is identical to an osteoblast-specific element-2 or acute myelogenous leukemia binding sequence in the mouse and rat osteocalcin genes, respectively. Overexpression of an acute myelogenous leukemia-1 repressor protein inhibited parathyroid hormone activation of the promoter, indicating a requirement of acute myelogenous leukemia-related factor(s) for this activity. Overexpression of c-Fos, c-Jun, osteoblast-specific factor-2, and core binding factor-β increased the response to parathyroid hormone of the wild type (−148) promoter but not with mutation of either or both the activator protein-1 and runt domain binding sites. In summary, we conclude that there is a cooperative interaction of acute myelogenous leukemia/polyomavirus enhancer-binding protein-2-related factor(s) binding to the runt domain binding site with members of the activator protein-1 transcription factor family binding to the activator protein-1 site in the rat collagenase-3 gene in response to parathyroid hormone in osteoblastic cells.

Isolation and Characterization of a Novel Coactivator Protein, NCoA-62, Involved in Vitamin D-mediated Transcription

The vitamin D receptor (VDR) forms a heterodimeric complex with retinoid X receptor (RXR) and binds to vitamin D-responsive promoter elements to regulate the transcription of specific genes or gene networks. The precise mechanism of transcriptional regulation by the VDR·RXR heterodimer is not well understood, but it may involve interactions of VDR·RXR with transcriptional coactivator or corepressor proteins. Here, a yeast two-hybrid strategy was used to isolate proteins that selectively interacted with VDR and other nuclear receptors. One cDNA clone designated NCoA-62, encoded a 62,000-Da protein that is highly related to BX42, a Drosophila melanogaster nuclear protein involved in ecdysone-stimulated gene expression. Yeast two-hybrid studies andin vitro protein-protein interaction assays using glutathione S-transferase fusion proteins demonstrated that NCoA-62 formed a direct protein-protein contact with the ligand binding domain of VDR. Coexpression of NCoA-62 in a vitamin D-responsive transient gene expression system augmented 1,25-dihydroxyvitamin D3-activated transcription, but it had little or no effect on basal transcription or gal4-VP16-activated transcription. NCoA-62 also interacted with retinoid receptors, and its expression enhanced retinoic acid-, estrogen-, and glucocorticoid-mediated gene expression. These data indicate that NCoA-62 may be classified into an emerging set of transcriptional coactivator proteins that function to facilitate vitamin D- and other nuclear receptor-mediated transcriptional pathways.

Stimulation of Extracellular Signal-regulated Kinases and Proliferation in Rat Osteoblastic Cells by Parathyroid Hormone Is Protein Kinase C-dependent

Parathyroid hormone (PTH) is known to have both catabolic and anabolic effects on bone. The dual functionality of PTH may stem from its ability to activate two signal transduction mechanisms: adenylate cyclase and phospholipase C. Here, we demonstrate that continuous treatment of UMR 106-01 and primary osteoblasts with PTH peptides, which selectively activate protein kinase C, results in significant increases in DNA synthesis. Given that ERKs are involved in cellular proliferation, we examined the regulation of ERKs in UMR 106-01 and primary rat osteoblasts following PTH treatment. We demonstrate that treatment of osteoblastic cells with very low concentrations of PTH (10− 12 to 10− 11 m) is sufficient for substantial increases in ERK activity. Treatment with PTH-(1–34) (10− 8 m), PTH-(1–31), or 8-bromo-cAMP failed to stimulate ERKs, whereas treatment with phorbol 12-myristate 13-acetate, serum, or PTH peptides lacking the N-terminal amino acids stimulated activity. Furthermore, the activation of ERKs was prevented by pretreatment of osteoblastic cells with inhibitors of protein kinase C (GF 109203X) and MEK (PD 98059). Treatment of UMR cells with epidermal growth factor (EGF), but not PTH, promoted tyrosine phosphorylation of the EGF receptor. Transient transfection of UMR cells with p21N17Ras did not block activation of ERKs following treatment with low concentrations of PTH. Thus, activation of ERKs and proliferation by PTH is protein kinase C-dependent, but stimulation occurs independently of the EGF receptor and Ras activation.

Parathyroid hormone induces c-fos promoter activity in osteoblastic cells through phosphorylated cAMP response element (CRE)-binding protein binding to the major CRE

Many parathyroid hormone (PTH)-mediated events in osteoblasts are thought to require immediate early gene expression. PTH induces the immediate early gene, c-fos, in this cell type through a cAMP-dependent pathway. The present work investigated the nuclear mechanisms involved in PTH regulation of c-fos in the osteoblastic cell line, UMR 106-01. By transiently transfecting c-fos promoter 5′ deletion constructs into UMR cells, we demonstrated that PTH induction of the c-fos promoter requires the major cAMP response element (CRE). Point mutations created in the major CRE within the largest construct inhibited both PTH-stimulated and basal expression. This element, therefore, performs concerted basal and PTH-responsive cis-acting functions. Gel retardation and Western blotting techniques revealed that CRE-binding protein (CREB) constitutively binds the major CRE but becomes phosphorylated at its cAMP-dependent protein kinase consensus recognition site following PTH treatment. CREB was functionally implicated in c-fos regulation by coexpressing a dominant CREB repressor, KCREB (killer CREB), with the c-fos promoter constructs. KCREB suppressed both basal and PTH-mediated c-fos induction. We conclude that PTH activates c-fos in osteoblasts through cAMP-dependent protein kinase-phosphorylated CREB interaction with the major CRE in the promoter region of the c-fos gene.

Collagenase-3 Binds to a Specific Receptor and Requires the Low Density Lipoprotein Receptor-related Protein for Internalization

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 125I-collagenase-3 binds specifically to two proteins (∼170 kDa and ∼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.

Collagenase‐3 (MMP‐13) and Integral Membrane Protein 2a (Itm2a) are Marker Genes of Chondrogenic/Osteoblastic Cells in Bone Formation: Sequential Temporal, and Spatial Expression of Itm2a, Alkaline Phosphatase, MMP‐13, and Osteocalcin in the Mouse

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.

Developmental regulation of collagenase-3 mRNA in normal, differentiating osteoblasts through the activator protein-1 and the runt domain binding sites

Collagenase-3 mRNA is initially detectable when osteoblasts cease proliferation, increasing during differentiation and mineralization. We showed that this developmental expression is due to an increase in collagenase-3 gene transcription. Mutation of either the activator protein-1 or the runt domain binding site decreased collagenase-3 promoter activity, demonstrating that these sites are responsible for collagenase-3 gene transcription. The activator protein-1 and runt domain binding sites bind members of the activator protein-1 and core-binding factor family of transcription factors, respectively. We identified core-binding factor a1 binding to the runt domain binding site and JunD in addition to a Fos-related antigen binding to the activator protein-1 site. Overexpression of both c-Fos and c-Jun in osteoblasts or core-binding factor a1 increased collagenase-3 promoter activity. Furthermore, overexpression of c-Fos, c-Jun, and core-binding factor a1 synergistically increased collagenase-3 promoter activity. Mutation of either the activator protein-1 or the runt domain binding site resulted in the inability of c-Fos and c-Jun or core-binding factor a1 to increase collagenase-3 promoter activity, suggesting that there is cooperative interaction between the sites and the proteins. Overexpression of Fra-2 and JunD repressed core-binding factor a1-induced collagenase-3 promoter activity. Our results suggest that members of the activator protein-1 and core-binding factor families, binding to the activator protein-1 and runt domain binding sites are responsible for the developmental regulation of collagenase-3 gene expression in osteoblasts.

Regulation of expression of collagenase-3 in normal, differentiating rat osteoblasts

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 postconfluence, 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 nonmineralizing medium expressed collagenase‐3 but did not show the developmental increase exhibited by cells switched to mineralization medium. Cells maintained in nonmineralizing 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. J. Cell. Physiol. 181:479–488, 1999.

Collagenase and Tissue Plasminogen Activator Production in Developing Rat Calvariae: Normal Progression Despite Fetal Exposure to Microgravity

Abstract. 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.