Increased osteoblastogenesis and decreased bone resorption protect against ovariectomy-induced bone loss in thrombospondin-2-null mice.

Although bone is composed primarily of extracellular matrix (ECM), the dynamic role that the ECM plays in regulating bone remodeling secondary to estrogen loss is relatively unexplored. Previous studies have shown that mice deficient in the matricellular protein thrombospondin-2 (TSP2-null) form excess endocortical bone; thus, we postulated that enhanced bone formation in TSP2-null mice could protect against ovariectomy (OVX)-induced bone loss. Wild-type (WT) OVX mice showed a significant loss of both midfemoral endocortical and proximal tibial trabecular bone, but OVX did not significantly alter TSP2-null bone. TSP2-null mice showed an increase in bone formation, as indicated by a 70% increase in serum osteocalcin two weeks post OVX and a two-fold increase in bone formation rate (BFR) five weeks post OVX as measured by dynamic histomorphometry. WT animals showed only a 20% increase in serum osteocalcin at two weeks and no change in BFR at five weeks. This increase in bone formation in TSP2-null OVX mice was accompanied by a three-fold increase in osteoprogenitor number. Although these results provide a partial explanation for the maintenance of bone geometry post-OVX, TSP2-null mice five weeks post-OVX also showed a significantly lower level of bone resorption than OVX WT mice, as determined by serum levels of the amino-terminal telopeptide of type I collagen (NTx). We conclude that the absence of TSP2 protects against OVX-induced bone loss by two complementary processes: increased formation and decreased resorption.

Analysis of muscle creatine kinase regulatory elements in recombinant adenoviral vectors.

Adenoviral gene transfer holds promise for gene therapy, but effective transduction of a large and distributed tissue such as muscle will almost certainly require systemic delivery. In this context, the use of muscle-specific regulatory elements such as the muscle creatine kinase (MCK) promoter and enhancer will avoid potentially harmful ectopic expression of transgenes. We describe here the development and testing of adenoviral vectors containing small, striated muscle-specific, highly active MCK expression cassettes. One of these regulatory elements (CK6) is less than 600 bp in length and is 12% as active as the CMV promoter/enhancer in muscle. A recombinant adenoviral vector containing this regulatory element retains very high muscle specificity, expressing 600-fold higher levels of transgene in muscle than in liver. Muscle-specific regulatory elements may also increase persistence of transduced muscle cells. Adenoviral transduction of dendritic cells has been shown to stimulate cytotoxic T-lymphocyte (CTL) responses directed against transgene epitopes. We show that human dendritic cells infected in vitro with MCK-containing adenoviruses do not express significant levels of transgene. Furthermore, while adenoviral vectors containing nonspecific promoters are normally cleared from muscle tissue within 1 month, we show that MCK-containing vectors express significant levels of transgene 4 months after intramuscular injection.

Hypercalcemia during the osteogenic phase after rat marrow ablation coincides with increased bone resorption assessed by the NTx marker.

Marrow ablation is a model of bone turnover in which the excavated tibial intramedullary cavity is rapidly and reproducibly filled by osteoblasts with new woven bone (days 6-8), which is then rapidly resorbed by osteoclasts (days 10-15). We showed previously (Magnuson et al., 1997) that marrow ablation induces a dramatic hypercalcemia and hypercalciuria in rats that unexpectedly peaked at the time of maximal osteogenesis and continued throughout the subsequent resorption phase. Based upon the amount of calcium mobilized and a peak of urinary hydroxyproline, we suggested that the hypercalcemia and hypercalciuria were due to increased systemic osteoclastic bone resorption induced by marrow ablation. We now apply a new enzyme-linked immunosorbent assay for rodent alpha(2)(I) N-telopeptide (NTx), a marker of bone resorption, to the marrow ablation model to demonstrate that excretion of NTx parallels that of calcium release in the operated control group. Specifically, maximal NTx/creatinine excretion coincides with the onset of hypercalcemia on days 7-8. A peak of NTx was also observed in methylprednisolone- and deflazacort-treated ablated animals. Analyses for urinary free deoxypyridinoline crosslink failed to detect a significant ablation-induced change in excretion. Interleukin 6 activity was increased in all operated control and glucocorticoid-treated groups after marrow ablation, whereas serum parathyroid hormone remained at presurgical levels in operated controls throughout the 15-day study period. The NTx results confirm that bilateral tibial marrow ablation induces a burst of extratibial bone resorption and hypercalcemia 7-8 days later. We have estimated that the osteogenic phase of the ablation model deposits 40 mg of calcium as hydroxyapatite crystals within the intramedullary cavity on days 6-8; this represents 33%-50% of the total blood calcium content of a young rat. We hypothesize that the size and rapidity of this demand for ionized calcium is met through an extratibial bone resorption pathway of osteoclast formation and activation that anticipates and fulfills this need, and that is initiated at the time of marrow ablation.

Osteoclasts generate cross-linked collagen N-telopeptides (NTx) but not free pyridinolines when cultured on human bone.

Urinary excretion of the cross-linked alpha 2(I) N-telopeptide (NTx) of type I collagen has proven in clinical studies to provide a highly responsive and specific index of bone resorption. In order to understand better the biological basis of the specificity and responsiveness of this marker, we examined whether osteoclasts cultured on human bone could generate immunoreactive NTx peptide. Mouse bone marrow cultures stimulated with 1,25 diliydroxyvitamin D3 (1,25(OH)2D3) and hydrocortisone to produce osteoclasts, were cocultured on human bone particles or dentin slices. Aliquots of culture medium were assayed for NTx by enzyme-linked immunosorbent assay (ELISA). NTx was detected in the medium 5 days after the addition of bone and continued to be produced linearly over the 14-day culture period. NTx production required attachment to the bone particles or dentin slices of mononuclear and multinuclear cells that stained for tartrate-resistant acid phosphatase. Surface area of resorbed dentin was highly correlated with medium NTx concentration (R2 = 0.84). Production of NTx was suppressed by the osteoclast inhibitors, calcitonin and alendronate, in a dose-dependent manner. Two other markers of bone resorption, hydroxylysyl pyridinoline and lysyl pyridinoline, were found in peptide linkage in the culture medium but not in free form; indicating that the osteoclasts had degraded the bone collagen to peptides but not to the free cross-linking amino acids.

Muscle gene E-box control elements. Evidence for quantitatively different transcriptional activities and the binding of distinct regulatory factors.

The muscle creatine kinase gene enhancer contains two regulatory elements (MCK-R and MCK-L) with the consensus E-box sequence (CAnnTG). A myocyte specific protein complex, MEF1, binds the MCK-R site. MEF1 contains several basic H-L-H myogenic determination factors (MDFs), each dimerized with ubiquitous members of the bH-L-H family (e.g. E12/E47). We now demonstrate that the ubiquitous bH-L-H factor E2-2 is a major component of the endogenous MCK-R site specific complex. Previous studies described the MCK-L site as a similar but low affinity MDF/bH-L-H heterodimer binding site. However, we find that the MCK-L site exhibits preferential binding of an unknown ubiquitous factor which contains neither E12/E47 nor E2-2, and that it exhibits differential transcriptional activity with muscle and non-muscle cells. The differential behavior of the MCK-L and MCK-R sites may be a general trait of E-box elements since one among several E-boxes in the MLC 1/3 enhancer also binds preferentially to the MCK-L factor. From our studies we now propose separate consensus sequences for MCK-R and MCK-L E-box types: AACAc/gc/gTGCa/t and GGa/cCANGTGGc/gNa/g. Our results suggest that while many muscle gene E-boxes are capable of binding the previously characterized spectrum of MDF/bH-L-H heterodimers in vitro, MCK-L type E-boxes probably bind qualitatively different factors in vivo.

MyoD is a sequence-specific DNA binding protein requiring a region of myc homology to bind to the muscle creatine kinase enhancer.

MyoD is a skeletal muscle-specific protein that is able to induce myogenesis in a wide variety of cell types. In this report, we show that MyoD is a DNA binding protein capable of specific interaction with two regions of the mouse muscle creatine kinase gene upstream enhancer, both of which are required for full muscle-specific enhancer activity. MyoD shares antigenicity and DNA binding specificity with MEF1, a myocyte-specific DNA binding factor. The contiguous basic and myc homology regions of MyoD that are necessary and sufficient for specific DNA interaction are the same regions of the protein required to convert 10T1/2 fibroblasts into muscle. These findings suggest that the biological activity of MyoD is mediated via its capacity for specific DNA interaction.

Interactions between the promoter and first intron are involved in transcriptional control of alpha 1(I) collagen gene expression.

The first intron of the human collagen alpha 1(I) gene contains several positively and negatively acting elements. We have studied the transcription of collagen-human growth hormone fusion genes, containing deletions and rearrangements of collagen intronic sequences, by transient transfection of chick tendon fibroblasts and NIH 3T3 cells. In chick tendon fibroblasts, but not in 3T3 cells, inversion of intronic sequences containing a previously studied 274-base-pair segment, A274, resulted in markedly reduced human growth hormone mRNA levels as determined by an RNase protection assay. This inhibitory effect was largely alleviated when deletions were introduced in the collagen promoter of plasmids containing negatively oriented intronic sequences. Evidence for interaction of the promoter with the intronic segment, A274, was obtained by gel mobility shift assays. We suggest that promoter-intron interactions, mediated by DNA-binding proteins, regulate collagen gene transcription. Inversion of intronic segments containing critical interactive elements might then lead to an altered geometry and reduced activity of a transcriptional complex in those cells with sufficiently high levels of appropriate transcription factors. We further suggest that the deleted promoter segment plays a key role in directing DNA interactions involved in transcriptional control.

Substitution of cysteine for glycine within the carboxyl-terminal telopeptide of the alpha 1 chain of type I collagen produces mild osteogenesis imperfecta.

We have characterized a mutation that produces mild, dominantly inherited osteogenesis imperfecta. Half of the alpha 1 (I) chains of type I collagen synthesized by cells from an affected individual contain a cysteine residue in the 196-residue carboxyl-terminal cyanogen bromide peptide of the triple-helical domain (Steinmann, B., Nicholls, A., and Pope, F. M. (1986) J. Biol. Chem. 261, 8958-8964). Unexpectedly, sequence determined from a proteolytic fragment of the alpha 1 (I) chain derived from procollagen molecules synthesized in the presence of both [3H]proline and [35S]cysteine indicated that the cysteine is located at the third residue carboxyl-terminal to the triple-helical domain, normally a glycine. The nucleotide sequence of a fragment amplified from genomic DNA confirmed the location of the cysteine residue and showed that the mutation was a single nucleotide change in one COL1A1 allele. This represents a new class of mutations, point mutations outside the triple-helical domain of the chains of type I collagen, that produce the osteogenesis imperfecta phenotype.

A growing family of collagens in articular cartilage: identification of 5 genetically distinct types.

Cartilage displays an extensive polymorphism of collagen types. All hyaline cartilages are known to contain type II collagen (90-95% of the collagen) and 2 other cartilage-specific minor collagens called types IX (or M) and XI (or 1 alpha 2 alpha 3 alpha). In the present work, the molecular heterogeneity of articular cartilage collagen was investigated in more detail. Two additional genetic types of collagen were found in the mature bovine tissue, types V and VI, each accounting for about 1-2% of the tissue dry weight. Type V, a close relative of type XI, apparently had accumulated after birth. Type VI is a short-helix, microfibrillar collagen of wide tissue distribution but unknown function. In understanding the physical changes in the collagen network of cartilage that are believed to herald the onset of cartilage degeneration in osteoarthrosis, all five of these collagen types deserve attention.