Heterodimeric bone morphogenetic proteins show enhanced activity in vitro and in vivo.

The bone morphogenetic proteins (BMPs), a subgroup of the TGF-beta gene super-family, are dimeric molecules involved in the growth, differentiation and repair of a wide variety of tissues. Based on the observation that several of the BMPs co-purify when isolated from bovine bone and that a pattern of co-localization exists during mouse embryogenesis, we co-expressed various combinations of BMPs in Chinese hamster ovary cells to test for possible heterodimer formation and activity. Transient co-expression of BMP-2 with either BMP-5, BMP-6 or BMP-7, or BMP-4 transiently co-expressed with BMP-7, resulted in more BMP activity than expression of any single BMP. Stable cell lines were then made in order to purify and characterize co-expressed BMPs in more detail. Co-expression of BMP-2 with BMP-7 yielded heterodimeric BMP-2/7 with a specific activity about 20-fold higher than BMP homodimers in an in vitro alkaline phosphatase induction assay. These heterodimers were also 5- to 10-fold more potent than BMP-2 in inducing cartilage and bone in an in vivo assay. Similar results were obtained with BMP-2/6 heterodimer. These experiments demonstrate the increased potency of several BMP heterodimers relative to BMP homodimers and support the hypothesis that such heterodimeric forms are likely to have natural biological functions.

Sequences in the coding region of clotting factor VIII act as dominant inhibitors of RNA accumulation and protein production.

A variety of retroviral vectors for transduction and expression of clotting factor VIII (FVIII) were constructed by using truncated forms of a FVIII cDNA lacking part or all of the nonessential B-domain sequences. Both the titer of virus and FVIII protein production from the vectors was about 2 orders of magnitude lower than the virus titer and protein production from identical retroviral vectors containing other cDNAs, including clotting factor IX. These decreases could be entirely explained by an observed 100-fold lower accumulation of vector RNAs containing the FVIII sequences in comparison to vectors containing other cDNA sequences. Deletion analysis of one of the FVIII vectors demonstrated that diffuse sequences within the FVIII coding region had a deleterious effect upon vector titer and RNA accumulation. One inhibitory signal could be localized to a 1.2-kb stretch of DNA, but further localization was not possible as additional size reduction abolished the activity. These results indicate that expression of FVIII is regulated by signals within FVIII coding sequence that result in decreased RNA accumulation and FVIII protein production. Alteration of these inhibitory signals to permit high-level FVIII production may be difficult due to the wide distribution of these signals within the coding region of the protein.

A PCR-based method for high stringency screening of DNA libraries.

A rapid method for cloning genomic DNA utilizing a PCR-based screening protocol is described. A murine genomic library in lambda phage was subdivided into 64 wells, each containing 1000 clones, and propagated in bacteria. Amplified phage from each of 8 wells across columns, and each of 8 wells down rows, were pooled. The pooled phage were screened for the presence of murine M-CSF DNA by PCR using specific oligonucleotide primers. A single well that contained an M-CSF genomic clone was identified by the synthesis of a PCR product of the correct size that hybridized to an internal M-CSF oligonucleotide probe. This well was subdivided into 64 wells, each containing approximately 30 individual phage, reamplified, and rescreened utilizing the same protocol. A positive well was then subdivided and amplified a third time starting with an average of 2 phage per well, and rescreened for M-CSF DNA by PCR. Phage from a PCR-positive well, now highly enriched for M-CSF DNA, were grown as individual plaques. PCR-screening of randomly picked plaques demonstrated that the majority contained an M-CSF genomic insert. This method obviates the more labor and time intensive method of plaque hybridization screening of DNA libraries, and is more stringent since three oligonucleotides (the two PCR primers, and the hybridization probe) are required to give a true positive signal. Similar methodology has also been used to clone a cDNA gene contained within a plasmid library.

Dexamethasone negatively regulates the activity of a chimeric dihydrofolate reductase/glucocorticoid receptor protein.

A chimeric gene was constructed encoding the entire murine dihydrofolate reductase (DHFR) protein with a carboxyl-terminal extension encompassing amino acids 494-795 of the rat glucocorticoid receptor (GR). The chimeric DHFR/GR gene encoded a functional DHFR protein, as measured by the ability to transform DHFR-deficient Chinese hamster ovary (CHO) cells to a DHFR-positive phenotype. The DHFR/GR protein bound [3H]dexamethasone with a similar affinity as wild-type GR. Selection of stable CHO transformants in increasing concentrations of methotrexate resulted in increased expression of DHFR/GR. Addition of dexamethasone, a synthetic glucocorticoid agonist, decreased the activity of the chimeric protein, as measured by colony formation in selective medium, binding of fluoresceinated methotrexate, and direct enzymatic assay for DHFR. Addition of RU486, a glucocorticoid antagonist, antagonized the effect of dexamethasone. In the absence of dexamethasone, the chimeric protein was primarily localized to the cytoplasm. In the presence of dexamethasone or RU486, DHFR/GR translocated into the nucleus. However, RU486 did not decrease DHFR activity, distinguishing subcellular location from functional activity. These results demonstrate that glucocorticoids negatively affect the function of DHFR/GR.

Bone morphogenetic protein-2 causes commitment and differentiation in C3H10T1/2 and 3T3 cells.

C3H10T1/2 cells are an established mesenchymal stem cell line which can differentiate into muscle, fat and cartilage cells when treated with azacytidine. Bone morphogenetic protein-2 (BMP-2) caused a dose dependent differentiation of these cells into fat, cartilage and bone cells-low concentrations favoring adipocytes and high concentrations chondrocytes and osteoblasts. The differentiated phenotypes were stable in the absence of BMP-2. Furthermore, the addition of other growth factors during the differentiation process altered the frequency of the differentiated colony formation. Transfection of the C3H10T1/2 cells with a BMP-2 cDNA also induced a phenotypic change from the parental fibroblast to adipocytes and osteoblasts. Our results in this model system indicate that a single protein factor can cause differentiation of a stem cell line to multiple phenotypes, that phenotypes induced can be regulated by factor concentration, and that other factors can also influence BMP-2 induced differentiation.

Expression and characterization of bone morphogenetic protein-2 in Chinese hamster ovary cells.

Bone is a dynamic tissue that responds to many factors including vitamin D, parathyroid hormone, estrogen, calcitonin, and bone morphogenetic proteins (BMPs). The ability to stimulate new bone growth would permit novel therapies for situations where bone mass has been lost due to accident or disease. Purified BMP-2, in conjunction with a suitable matrix, is sufficient to stimulate the synthesis of new bone (Wang et al., 1990). We have expressed recombinant human BMP-2 at high levels in Chinese hamster ovary cells using methotrexate-mediated gene amplification. Several forms of BMP-2 are secreted from CHO cells: (1) an amino-terminal propeptide of 40-45 kDa, (23) a mature active 30 kDa homodimer consisting of 18-22 kDa subunits, and (3) a small amount of uncleaved 60 kDa precursor protein. The mature, active protein is predominantly a 30 kDa homodimer consisting of subspecies of 18 and 22 kDa which differ by proteolytic processing at their amino termini. Mature BMP-2 and propeptide contain high mannose and complex N-linked oligosaccharides, respectively. The molar amount of secreted, processed propeptide is approximately 5-fold higher than mature BMP-2 in conditioned medium. BMP-2 associates with both the extracellular matrix and the surface of CHO cells, which may in part account for the unequal levels of extracellular propeptide and mature forms of the molecule in the conditioned medium. Recombinant BMP-2 can be expressed in sufficient quantities to assess its therapeutic potential for bone regeneration.

Expression and characterization of TCA3: a murine inflammatory protein.

TCA3 is a cDNA originally isolated from activated T cells. Transcription of this gene has been shown to correlate with Ag-induced cellular activation of both T cells and mast cells. Based on the predicted amino acid sequence encoded by the cDNA, we previously proposed that TCA3 represents a cytokine. In this report we have used rDNA technology to express TCA3 in two mammalian cell lines. In both cases, TCA3 was expressed as a secreted molecule with an apparent molecular mass of 16 kDa. Digestion of the (rTCA3) with the enzyme N-glycanase revealed that approximately 8 kDa is caused by N-linked glycosylation. Intradermal injection of rTCA3 into mouse footpads resulted in a rapid swelling response. The sites of injection were characterized histologically by a local accumulation of neutrophils. These findings are discussed with particular attention to a family of related proteins, some of whose members also have inflammatory properties.

Recombinant human bone morphogenetic protein induces bone formation.

We have purified and characterized active recombinant human bone morphogenetic protein (BMP) 2A. Implantation of the recombinant protein in rats showed that a single BMP can induce bone formation in vivo. A dose-response and time-course study using the rat ectopic bone formation assay revealed that implantation of 0.5-115 micrograms of partially purified recombinant human BMP-2A resulted in cartilage by day 7 and bone formation by day 14. The time at which bone formation occurred was dependent on the amount of BMP-2A implanted; at high doses bone formation could be observed at 5 days. The cartilage- and bone-inductive activity of the recombinant BMP-2A is histologically indistinguishable from that of bone extracts. Thus, recombinant BMP-2A has therapeutic potential to promote de novo bone formation in humans.

Retroviral-mediated transfer and amplification of a functional human factor VIII gene.

Hemophilia A results from a deficiency in factor VII (FVIII), a cofactor in the intrinsic pathway of blood coagulation. As an approach toward genetic therapy of this disease, we constructed a retroviral vector encoding human FVIII and a selectable and amplifiable genetic marker, human adenosine deaminase (Ada). A retrovirus packaging line was transfected with this vector and stable transformants were selected for Ada expression. Isolated transformants produced both FVIII activity in the conditioned medium and retrovirus capable of transferring the Ada selectable marker and FVIII expression to the mouse 3T3 fibroblasts. Selection of virus-producer cell lines for increasing levels of Ada expression yielded a 20-fold increase in both FVIII expression and viral titer. Similarly, selection of infected 3T3 fibroblasts for Ada gene amplification yielded a 20-fold increase in FVIII expression. The results demonstrate the feasibility of retrovirus-mediated transfer of human FVIII, and also the utility of selection for gene amplification to increase retrovirus titers in producer cell lines as well as expression levels in infected cells.

Highly inducible expression from vectors containing multiple GRE's in CHO cells overexpressing the glucocorticoid receptor.

A conditional glucocorticoid-responsive expression vector system is described for highly inducible expression of heterologous genes in mammalian cells. This host-vector system requires high level expression of the glucocorticoid receptor (GR) protein in the host cell and multiple copies of the receptor binding site within the expression vector. Transfection and selection of Chinese hamster ovary cells with expression vectors encoding the rat GR yielded cell lines which express functional receptor at high levels. Insertion of multiple copies of the MMTV enhancer (glucocorticoid responsive element, GRE) into an Adenovirus major late promoter (AdMLP) based expression vector yielded greater than 1000-fold inducible expression by dexamethasone (dex) in transient DNA transfection assays. The induced expression level was 7-fold greater than that obtained with an AdMLP based vector containing an SV40 enhancer, but lacking GRE's. Vectors containing the SV40 enhancer in combination with multiple GRE's exhibited elevated basal expression in the absence of dex, but retained inducibility in both transient assays and after integration and amplification in the CHO genome. This expression system should be of general utility for studying gene regulation and for expressing heterologous genes in a regulatable fashion.

Effect of von Willebrand factor coexpression on the synthesis and secretion of factor VIII in Chinese hamster ovary cells.

In plasma, antihemophilic factor (factor VIII) exists as a 200-kilodalton heavy-chain polypeptide in a metal ion association with an 80-kilodalton light-chain polypeptide. This complex is bound by hydrophobic and hydrophilic interactions to a large multimeric glycoprotein, von Willebrand factor (vWF). Accumulation of secreted human factor VIII activity expressed in Chinese hamster ovary cells requires the addition of serum in the growth medium, which provides vWF. Here we report that coexpression of vWF with factor VIII in Chinese hamster ovary cells resulted in increased stable accumulation of factor VIII activity in the absence of serum in the growth medium. In the coexpressing cells, the vWF cDNA transcription unit was transcribed to yield mRNA which was efficiently translated. vWF was properly processed and secreted to yield disulfide-bonded high-molecular-weight multimers similar to those observed in vWF secreted from human endothelial cells. Nuclear run-on assays showed that the factor VIII gene was transcribed at a level similar to that of the vWF gene, but the mRNA did not accumulate to high levels in the cytoplasm. In addition, although the translation efficiency of the factor VIII mRNA was similar to that of vWF, the processing and secretion of the factor VIII primary translation product was dramatically reduced compared with vWF. These results demonstrate that in Chinese hamster ovary cells both factor VIII mRNA accumulation and the processing and secretion of the primary factor VIII translation product are inefficient processes.

Superinduction of cytochrome P1-450 gene transcription by inhibition of protein synthesis in wild type and variant mouse hepatoma cells.

Inhibition of protein synthesis superinduces transcription of the cytochrome P1-450 gene in Hepa 1c1c7 mouse hepatoma cells. The superinduced transcription rate is 10-15-fold higher than the maximal rate of transcription induced by the known inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alone. Superinduction is maximal within 30-40 min and remains maximal for at least 90 min. Cytochrome P1-450 mRNA is the same length in TCDD-induced and superinduced cells. Superinduction does not occur in variant cells in which TCDD-receptor complexes bind weakly to nuclei and which do not transcribe the cytochrome P1-450 gene in response to TCDD. Inhibition of protein synthesis does not alter several properties of TCDD-receptor complexes. The results imply that a second control mechanism modulates the action of the TCDD-receptor complex in regulating cytochrome P1-450 gene transcription.

Biochemical and genetic analysis of variant mouse hepatoma cells which overtranscribe the cytochrome P1-450 gene in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Using the fluorescence-activated cell sorter, we have isolated a population of variant mouse hepatoma cells which have a markedly increased ability to metabolize benzo(a)pyrene. Compared with wild-type (Hepa 1c1c7) cells, the variant cells exhibit increased aryl hydrocarbon hydroxylase activity and increased responsiveness of the aryl hydrocarbon hydroxylase induction mechanism to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Cell fusion experiments indicate that the variant phenotype is co-dominant with respect to wild-type. Filter hybridization analyses indicate that increased accumulation of cytochrome P1-450-specific mRNA accounts for the overproduction of aryl hydrocarbon hydroxylase activity. Measurements of RNA synthesis in isolated nuclei reveal that the variants exhibit an increased rate of transcription of the cytochrome P1-450 gene in response to TCDD. The variant cells contain no detectable alteration in their TCDD receptors, nor is the cytochrome P1-450 gene amplified in the variants. Filter hybridization analyses of restriction endonuclease-digested DNA indicate that the variant cytochrome P1-450 gene is relatively undermethylated, compared with the wild-type gene. We conclude that the variant cells contain an altered cis-acting genomic element(s) which regulates the expression of the cytochrome P1-450 gene.

Regulation of cytochrome P1-450 gene transcription by 2,3,7, 8-tetrachlorodibenzo-p-dioxin in wild type and variant mouse hepatoma cells.

Using RNA synthesized in isolated nuclei, we have measured the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the rate of synthesis of cytochrome P1-450 mRNA in wild type mouse hepatoma (Hepa 1c1c7) cells and in variant (BPrc1) cells, which fail to accumulate the TCDD-receptor complex within the nucleus. In wild type cells, TCDD induces a 20-fold increase in the rate of synthesis of cytochrome P1-450 mRNA within 30 min. This effect persists for at least 18 h. In contrast, TCDD has no effect on cytochrome P1-450 mRNA synthesis in the variant cells. The results demonstrate that TCDD increases the rate of transcription of the cytochrome P1-450 gene and suggest that transcription requires nuclear localization of the TCDD-receptor complex.

Induction of mRNA specific for cytochrome P1-450 in wild type and variant mouse hepatoma cells.

We have used a cDNA probe specific for an aromatic hydrocarbon-inducible form of cytochrome P-450 to analyze the accumulation of enzyme-specific mRNA in wild type Hepa 1c1c7 cells and in variant cells defective in aryl hydrocarbon hydroxylase induction. In wild type cells, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces a 25-50-fold increase in the steady state concentration of cytochrome P1-450-specific RNA. This RNA binds to oligo(dT)-cellulose and migrates in agarose gels as a single species of about 2900 nucleotides. Dot hybridization analyses of total RNA indicate that half-maximal induction of P1-450 mRNA occurs at about 10 pM TCDD; maximal induction occurs at about 100 pM TCDD. Parallel increases occur in aryl hydrocarbon hydroxylase activity. RNA induction precedes aryl hydrocarbon hydroxylase induction by 2-4 h. Kinetic analyses of mRNA induction in response to submaximal concentrations of TCDD reveal no change in the degradation of P1-450 mRNA during induction. We detect an increase in P1-450 mRNA 30 min after TCDD administration. Full accumulation of mRNA occurs in cells in which protein synthesis is inhibited by 95-97%. Variant cells with decreased TCDD receptors exhibit decreased basal and inducible levels of P1-450 mRNA. Variant cells with a defect in nuclear localization of the inducer-receptor complex exhibit virtually no basal or inducible levels of P1-450 mRNA. Hybrid cells formed by fusing these different variants contain wild type basal and inducible levels of P1-450 mRNA. We conclude that expression of the cytochrome P1-450 gene is under transcriptional control and requires nuclear localization of the TCDD receptor.