Adenovirus-mediated BMP2 expression in human bone marrow stromal cells.

Recombinant adenoviral vectors have been shown to be potential new tools for a variety of musculoskeletal defects. Much emphasis in the field of orthopedic research has been placed on developing systems for the production of bone. This study aims to determine the necessary conditions for sustained production of high levels of active bone morphogenetic protein 2 (BMP2) using a recombinant adenovirus type 5 (Ad5BMP2) capable of eliciting BMP2 synthesis upon infection and to evaluate the consequences for osteoprogenitor cells. The results indicate that high levels (144 ng/ml) of BMP2 can be produced in non-osteoprogenitor cells (A549 cell line) by this method and the resultant protein appears to be three times more biologically active than the recombinant protein. Surprisingly, similar levels of BMP2 expression could not be achieved after transduction with Ad5BMP2 of either human bone marrow stromal cells or the mouse bone marrow stromal cell line W20-17. However, human bone marrow stromal cells cultured with 1 microM dexamethasone for four days, or further stimulated to become osteoblast-like cells with 50 microg/ml ascorbic acid, produced high levels of BMP2 upon Ad5BMP2 infection as compared to the undifferentiated cells. The increased production of BMP2 in adenovirus transduced cells following exposure to 1 microM dexamethasone was reduced if the cells were not given 50 microg/ml ascorbic acid. When bone marrow stromal cells were allowed to become confluent in culture prior to differentiation, BMP2 production in response to Ad5BMP2 infection was lost entirely. Furthermore, the increase in BMP2 synthesis seen during differentiation was greatly decreased when Ad5BMP2 was administered prior to dexamethasone treatment. In short, the efficiency of adenovirus mediated expression of BMP2 in bone marrow stromal cells appears to be dependent on the differentiation state of these cells.

Characterization of bone morphogenetic protein 4 receptor in fibrodysplasia ossificans progressiva.

Bone morphogenetic protein 4, a potent osteogenic morphogen, has been implicated in fibrodysplasia ossificans progressiva because it is uniquely overexpressed in lymphoblastoid cells and preosseous fibroproliferative lesional cells of patients with fibrodysplasia ossificans progressiva. Bone morphogenetic protein 4 signals through a heteromeric complex of serine/ threonine kinase receptors (type I and type II) on the surface of responding cells. Semi-quantitative competitive reverse transcription polymerase chain reaction was used to quantitate steady state levels of messenger ribonucleic acid expression for bone morphogenetic protein 4 and the bone morphogenetic protein receptors. These data confirmed the previous finding of elevated steady state levels of bone morphogenetic protein 4 messenger ribonucleic acid in lymphoblastoid cell lines of affected individuals in a family that exhibited autosomal dominant inheritance of fibrodysplasia ossificans progressiva. There were no differences in the steady state levels of messenger ribonucleic acid for either the Type I or Type II bone morphogenetic protein 4 receptors between affected and unaffected individuals in that same family. The presence of bone morphogenetic protein 4 receptor messenger ribonucleic acid in fibrodysplasia ossificans progressiva lesional tissue and unaffected muscle tissue and demonstrates the deregulation of bone morphogenetic protein 4 messenger ribonucleic acid in fibrodysplasia ossificans progressiva. These data support the hypothesis that the molecular basis of bone morphogenetic protein 4 signaling is abnormal in fibrodysplasia ossificans progressiva.

Embryonic overexpression of the c-Fos protooncogene. A murine stem cell chimera applicable to the study of fibrodysplasia ossificans progressiva in humans.

Murine embryonic overexpression of the c-fos protooncogene leads to early postnatal heterotopic chondrogenesis and osteogenesis with phenotypic features similar to those seen in children who have the disabling heritable disease fibrodysplasia ossificans progressiva. The overexpression of Fos in embryonic stem cell chimeras leads to heterotopic endochondral osteogenesis at least in part through a bone morphogenetic protein 4 mediated signal transduction pathway. In contrast, early fibrodysplasia ossificans progressiva lesions express abundant bone morphogenetic protein 4, without abundant expression of c-Fos, suggesting that the primary molecular defect in fibrodysplasia ossificans progressiva may be independent of the sustained Fos effects on chondrogenesis and osteogenesis. Comparisons of the clinical, molecular, and pathogenetic features of the c-Fos embryonic stem cell chimeras with those of fibrodysplasia ossificans progressiva provide insight into the earliest events in the molecular pathogenesis of genetically induced heterotopic chondrogenesis and osteogenesis. The relevance of the c-Fos embryonic stem cell chimera to the study of the currently untreatable human disease fibrodysplasia ossificans progressiva demonstrates the power of using embryonic stem cell technology for generating gain of function mutations in the study of human bone disease.

Parathyroid hormone regulates the expression of the receptor protein tyrosine phosphatase, OST-PTP, in rat osteoblast-like cells.

Bone remodeling requires regulated tyrosine phosphorylation mediated by specific protein tyrosine kinases, such as c-src and c-fms, and to date, unknown protein tyrosine phosphatases (PTPs). We previously reported the isolation of a novel bone-specific receptor PTP, named osteotesticular PTP (OST-PTP), which is regulated during osteoblast differentiation and after exposure to PTH. To determine the relevance of this PTH regulation, we characterized the PTH-induced increase in OST-PTP messenger RNA (mRNA) in UMR 106 cells in comparison with PTH effects on a related receptor PTP and a PTH regulated gene, rat collagenase. Treatment of cells with rat PTH 1-34 (rPTH) resulted in a dramatic concentration and time-dependent increase in OST-PTP mRNA with a threshold at 4 h (= or < 1nM rPTH) and maximal response of 6- 10-fold above control levels at 8 h (100 nM rPTH). An increase in collagenase mRNA was detectable 2 h earlier at 100 pM rPTH with a maximal response at least 5-fold greater than that observed for OST- PTP. Levels of mRNA for the structurally similar PTP, rat leucocyte antigen-related molecule, were unaffected by rPTH treatment. Administration of cycloheximide (5-100 microM) abolished the OST-PTP and collagenase responses to PTH. The cAMP analogs, CPT-cAMP (0.01-1mM; 8 h) or Sp-cAMP (0.1 and 0.5 mM) were equal or greater in their effectiveness to enhance both OST-PTP and collagenase mRNA as compared with rPTH. In contrast, phorbol esters, calcium ionophore, bovine PTH (3-34), or human PTHrP (7-34) had no effect on either transcript. Interestingly, 36 h of pretreatment of cells with epidermal growth factor (10 ng/ml), a growth factor known to modulate PTH's actions, resulted in a significant decrease in the abundance of OST-PTP mRNA after rPTH exposure. These studies suggest that regulation of OST-PTP mRNA is a secondary response to PTH stimulation that is dependent on protein synthesis and that may be primarily by activation of the protein kinase A pathway. This specific modulation of a bone receptor PTP may prove to be a critical component in the PTH modulation of osteoblast function.

Identification of a hormonally regulated protein tyrosine phosphatase associated with bone and testicular differentiation.

Absence of the tyrosine kinase activity of c-src and c-fms results in impairment of bone remodeling. Such dysfunction underscores the importance of tyrosine phosphorylation, yet the role of protein tyrosine phosphatases in bone metabolism remains unexamined. We have isolated the cDNA for a novel receptor-like tyrosine phosphatase expressed in bone and testis named osteotesticular protein tyrosine phosphatase (OST-PTP). The deduced 1711-residue protein possesses an extracellular domain with 10 fibronectin type III repeats and a cytoplasmic region with two catalytic domains. In primary rat osteoblasts, the 5.8-kilobase OST-PTP transcript is up-regulated in differentiating cultures and down-regulated in late stage mineralizing cultures. In addition, a presumed alternate transcript of 4.8-5.0 kilobases, which may lack PTP domains, is present in proliferating osteoblasts, but not detectable at other stages. Parathyroid hormone, a modulator of bone function, as well as cyclic AMP analogues, increase OST-PTP mRNA 5-8-fold in UMR 106 cells. In situ hybridization of adult rat testis revealed stage-specific expression of OST-PTP. OST-PTP may function in signaling pathways during bone remodeling, as well as serve a broader role in cell interactions associated with differentiation in bone and testis.

Purification and characterization of eukaryotic initiation factor (eIF)-2 alpha kinases from Ehrlich ascites tumor cells.

A major mechanism of regulation of mammalian protein synthesis initiation is accomplished by the phosphorylation of the alpha subunit of eukaryotic initiation factor (eIF) 2. This modification inhibits the activity of another initiation factor, guanine nucleotide exchange factor, preventing conversion of eIF-2.GDP to eIF-2.GTP and hence binding of initiator tRNA and formation of ternary complex (eIF-2.GTP.Met-tRNAf). Inhibition of protein synthesis and phosphorylation of eIF-2 occurs in Ehrlich cells when they are amino acid- or serum-deprived or heat-shocked as well as in other nucleated cells under similar conditions. This paper describes the purification of two eIF-2 alpha kinases from Ehrlich cells. Unlike the two well characterized eIF-2 alpha kinases, HRI (heme-regulated inhibitor from reticulocytes) and P68 (double-stranded RNA-dependent kinase found in interferon-treated cells), the Ehrlich cell kinases do not appear to autophosphorylate. The two kinases chromatograph differently on DEAE-cellulose and Mono Q. Furthermore, their Michaelis constants (Km values) for ATP are different. Both kinases can inhibit purified guanine nucleotide exchange factor (GEF) from stimulating ternary complex formation. However, only one kinase inhibits reticulocyte lysate cell free protein synthesis. The other kinase co-purifies with a factor that suppresses inhibition of protein synthesis in reticulocyte lysates by eIF-2 alpha kinases. This suppressing activity is probably guanine nucleotide exchange factor.