Riggs, Lawrence B.

Abstract Serum bone Gla-prolein (BGP), also called osteocalcin, is a specific and sensitive measure of bone turnover in a variety of metabolic bone disorders. Although some BGP diffuses into the circulation after synthesis by osteoblasts, most is incorporated into bone matrix where it remains until bone is resorbed. Thus, serum BGP could reflect bone formation, bone resorption, or a combination of both. The relationship of serum BGP to the components of bone turnover was evaluated in 18 normal women (mean age 48 yr; range 30-70) who received a continuous 24-h intravenous infusion of the 1-34 synthetic fragment of bovine parathyroid hormone. Mean ± SE for urinary hydroxyproline excretion, an index of bone resorption, increased (from 22.7 ± 2.2 to 38.5 ± 3.7 μg/100 ml glomerular filtrate [GF], p < .001), whereas levels of serum alkaline phosphatase, an index of bone formation, were unchanged (from 20 ± 1 to 20 ± 1 U/liter, NS). Despite the increase in bone resorption, levels of serum BGP decreased (from 8.8 ± 0.8 to 6.8 ng/dl, p < .001). The data suggest that circulating levels of BGP are a measure of bone formation but, at least in subjects with normal renal function, not a measure of bone resorption. Presumably BGP in bone matrix is degraded during osteoclastic resorption into fragments that either are not recognized by an antiserum raised against the native molecule or are rapidly cleared from the circulation.

Abstract Although levels of serum immunoreactive parathyroid hormone (iPTH) increase with age in women, this could be caused by retention of non-biologically active PTH fragments by the aging kidney. In 102 normal women, aged 30 to 89 yr, serum iPTH increased with age by 58% (r = 0.33, p < 0.001) with antiserum GP-1M (which has midmolecule specificity) and 43% (r = 0.32, p < 0.001) with antiserum CH-12M (which may have whole molecule specificity); urinary cAMP/GFR excretion increased by 29% (r = 0.22, p < 0.05). The results of these assays were validated by comparison with serum levels of biologically active PTH (BioPTH) in immunoextracts of serum followed by renal adenylate cyclase assay in a selected subgroup of 25 of the women. Serum BioPTH correlated with serum iPTH assessed by antiserum GP-1M (r = 0.48, p < 0.05) and antiserum CH-12M (r = 0.48, p < 0.05) but not with urinary cAMP. The data are consistent with an increase of parathyroid function with aging: clearly, we do not find decreased parathyroid function as would be expected if age-related bone loss was not mediated, in part, by PTH.

Abstract To determine if bone cells produce interleukin-1β (IL-1β), a potent bone resorption-stimulating agent, we studied well-characterized, nearly homogeneous cultures of normal human osteoblast-like (hOB) cells. With four strains of such cells, vehicle-treated cultures produced minimal IL-1β (mean ± SEM, 1.3 ± 0.3 pg/ml per 106 cells per 24 h) and showed dose-dependent (r = 0.99) increases to 2.2 ± 0.7, 5.0 ± 0.9, or 17.8 ± 6.7 pg/ml, respectively, after treatment with lipopolysaccharide (LPS) at 3, 10, or 30 μg/ml (for increases after 10 and 30 μg/ml treatments, P < 0.05). After treatment with tumor necrosis factor α (TNF-α at 10 U/ ml, IL-1β increased to 16.2 * 3.7 pg/ml (P < 0.05). Neither 17β-estradiol nor bovine parathyroid hormone(1–34) (each at 10 nM), alone or in combination with LPS or TNF-α, affected IL-1β release. Northern blot analysis of total cellular RNA preparation revealed a single hybridization band at 1.9 kb when probed with a partially deleted cDNA for human IL-1β. The steady-state IL-1β mRNA levels showed a significant increase with LPS treatment and a lesser increase with TNF-α treatment in hOB cells. Moreover, TNF-α produced an even greater increase in IL-1 mRNA in HOBIT cells, a well-differentiated clonal cell line derived from normal hOB cells transfected with the SV40 large T antigen. We conclude that human cells of the osteoblast lineage produce IL-Iβ in response to well-recognized stimuli for IL-1 release from responsive tissue. Therefore, IL-1 may play an important role in the local regulation of bone remodeling.

Abstract A new bone cell line was established by transfecting normal adult human osteoblast-like (hOB) cells, derived from a 68-year-old woman, with the plasmid pSV3 neo. The plasmid included coding sequences and promotors for the large and small T antigens of the SV40 virus as well as resistance to the antibiotics neomycin and G418. A single antibiotic-resistant colony was located and cloned. Large tumor antigen production in the clonal cell line was confirmed by indirect immunofluorescence study. Treatment with 1,25-dihydroxy-vitamin D3 increased steady-state concentrations of protein and mRNA for osteocalcin and for alkaline phosphatase. Northern blot analyses also demonstrated the presence of mRNAs for α(I)-procollagen, osteopontin 1a, transforming growth factor β, and interleukin-1β. The plasma membrane calcium pump and osteonectin were identified by immunocytochemical analysis. These cells produced a matrix that mineralized when β-glycerophosphate was added to their cultures. As assessed by functional receptor assays, both estrogen and androgen receptors were present and functional, although at low concentrations. Treatment with parathyroid hormone did not stimulate adenylate cyclase activity. Thus, these cells are a well-differentiated, steroid-responsive clonal cell line that closely approximates the phenotype of the mature osteoblast. They should serve as an excellent model for the study of osteoblast biology.

Abstract Mutations in one of the two genes encoding type I procollagen (COL1A1 and COL1A2) are frequently the cause of osteogenesis imperfecta (OI), a disorder characterized by brittle bones. Here we tested whether patients with low bone density also have mutations in these genes. The 26 patients studied had no apparent metabolic bone disease, but most had a positive family history of osteopenia or osteoporosis. Although a diagnosis of OI was considered by the clinician in some cases, the clinical criteria for OI were not satisfied. Our strategy for mutation analysis consisted of PCR amplification of cDNA made to fibroblast mRNA using primers specific for the coding regions of COL1A1 and COL1A2. The PCR products were then sequenced directly with primers located within each PCR product. We found that 3 of 26 patients had mutations that altered the encoded amino acid. One mutation, at position α2(I)-661 has been reported (Spotila et al. 1991 Proc Natl Acad Sci USA PNAS 88: 5423). The other 2 patients, who were not related to each other, had a mutation that altered the proline codon at α1(I)-27 to alanine. This mutation was not found in 81 normal individuals or in 37 additional osteopenic individuals. However, its effect on the biologic function of type I collagen, as well as its role in osteopenia, is uncertain. In addition to the two mutations, we found a polymorphism in codon α2(I)-459. Although this polymorphism involved an amino acid substitution, it was present with equal frequency in the patient and the normal population. By analyzing this and previously reported neutral sequence variants in the COL1A2 gene, we determined that all patients expressed both alleles of the COL1A2 gene. The 12 patients who were heterozygous for a COL1A1 neutral sequence variant also expressed both alleles. Here we present all PCR primer and sequencing primer information. The results suggest that surveying a larger group of similarly selected individuals may reveal additional mutations in the COL1A1 or COL1A2 genes.

Abstract Insulin-like growth factor binding protein 4 (IGFBP-4) is secreted by normal human osteoblast-like cells (hOB) and is a potent inhibitor of insulin-like growth factor (IGF) action in vitro. In previous studies, IGF treatment of hOB in culture led to markedly reduced medium levels of IGFBP-4 as detected by western ligand blotting. In the present study, incubation of hOB-conditioned medium (hOB-CM) with IGF under cell-free conditions resulted in a similar loss of IGFBP-4. Both IGF-I and IGF-II were capable of inducing a decrease in IGFBP-4; however, IGF-II was more effective. When the six characterized IGFBP were added to hOB-CM, only IGFBP-4 disappeared in response to IGF-II addition. This IGF-regulated loss of IGFBP-4 was inhibited by metalloproteinase inhibitors and appeared to be due to a proteinase that cleaved IGFBP-4 in 18 and 14 kD fragments identified by western immunoblotting. Conditioned media from eight of eight different donor hOB lines tested exhibited IGFBP-4 proteinase activity. To assess the biologic consequences of IGF-II-induced IGFBP-4 proteolysis, we treated hOB with IGF-II for 5 h, which decreased medium IGFBP-4 by 70%, and then measured IGF-I and insulin stimulation of [3H]thymidine incorporation. IGF-II itself was not mitogenic and had no effect on insulin-stimulated [3H]thymidine incorporation. However, pretreatment of cultured hOB with IGF-II enhanced IGF-I-stimulated [3H]thymidine incorporation threefold. In conclusion, these data indicate that (1) regulation of IGFBP-4 availability can occur via an IGF-dependent, IGFBP-4 proteinase secreted by normal hOB, and (2) IGF-II-induced IGFBP-4 proteolysis is associated with enhanced hOB response to IGF-I. The IGFBP-4/IGFBP-4 proteinase system may be involved in local regulation of IGF action in bone.

Abstract Bone mass and its mineral content are under genetic control. The vitamin D receptor (VDR) gene has been shown to be a major locus for genetic effects on bone mineral density (BMD), and polymorphisms in this gene accounted for a large proportion of genetic variance in BMD in an Australian population. In this study, we investigated whether similar associations are present in a North American population. We studied 139 normal healthy women (age 53.2 ± 14.5, mean ± SD) and 43 severely osteoporotic postmenopausal women (age 65.8 ± 5.9). In the 127 of them with complete genetic studies, the distribution of genotypes, determined by polymerase chain reaction on leukocyte DNA samples, agreed closely with that in the Australian population. BMD was strongly related to age and weight, and, thus was adjusted for these parameters prior to genetic analysis. We found that age modulated the effect of VDR genotypes on femoral neck BMD (FN-BMD) (TaqI, p = 0.036;BsmI, p = 0.118; ApaI, p = 0.041) such that the effect of genotype was greatest among younger (premenopausal) women and declined with age so that there was no discernible difference by age 70. Among the younger women, a high FN-BMD was associated with the TT (or aa or bb) genotype while low FN-BMD was associated with the tt (or AA or BB) genotype. In osteoporotic women, although the difference in FN-BMD between alternate TaqI homozygotes (TT vs. tt) was 0.13 g/cm2 (or about 18%), the overall difference among the genotype groups was not significant (TaqI, p = 0.13; BsmI, p = 0.21; ApaI, p = 0.09), possibly due to the small sample of osteoporotic women. There was no relationship of VDR genotype to lumbar spine BMD. We conclude that the VDR gene modulates differences in BMD in premenopausal women. Subsequent interaction of the gene and environmental factors and the role of VDR genotypes in predisposing to osteoporosis needs further study.

Abstract Insulin-like growth factors I (IGF-I) and II (IGF-II) are anabolic for osteoblastic cells. Although expression of IGF-I and IGF-II mRNA has been demonstrated in rodent osteoblastic cells, little is known about IGF gene expression in human osteoblastic cell models. In this study we characterized IGF-I and -II mRNA expression in (1) normal human osteoblast-like (hOB) cells, (2) a simian virus 40 immortalized hOB (HOBIT) cell line, and (3) human osteosarcoma cell lines SaOS-2, TE-85, MG-63, and U-2. Since cross-hybridization of IGF cDNA probes with ribosomal RNA obscures detection of some of the multiple IGF transcripts in human cells, we replaced Northern analysis with the more specific ribonuclease protection assay (RPA). We also used the reverse transcriptase-polymerase chain reaction (RT-PCR) to assess whether mRNAs were present at trace levels. IGF-I mRNA expression was consistently observed in normal hOB cells only and by both RT-PCR and RPA. Among IGF-I transcript variants, Ea IGF-I mRNA was more abundant than the Eb mRNA in normal hOB cells. Trace levels of IGF-I mRNA were variably detected in SaOS-2 and U-2 osteosarcoma cells when RT-PCR was performed, but we found no IGF-I mRNA in HOBIT, TE-85, or MG-63 cells. IGF-II mRNA was expressed in normal hOB, HOBIT, TE-85, and U-2 cells as assessed by either method. Trace levels of IGF-II mRNA were observed only in one of three SaOS-2 cell preparations and only by RT-PCR. IGF-II mRNA was absent in MG-63 cells. Thus, our data indicate that (1) normal hOB cells express both IGF-I and IGF-II mRNA and (2) transformation of human osteoblastic cells may alter IGF gene expression. These results provide a basis for selecting appropriate cell models for the study of IGFs in human bone.

Abstract We report the establishment of a human fetal osteoblast cell line derived from biopsies obtained from a spontaneous miscarriage. Primary cultures isolated from fetal tissue were transfected with a gene coding for a temperature-sensitive mutant (tsA58) of SV40 large T antigen along with a gene coding for neomycin (G418) resistance. Individual neomycin resistant colonies were screened for alkaline phosphatase (AP)-specific staining. The clone with the highest AP level, hFOB 1.19, was examined further for other osteoblast phenotypic markers. Incubation of hFOB cells at the permissive temperature (33.5°C) resulted in rapid cell division, whereas little or no cell division occurred at the restrictive temperature (39.5°C). Both AP activity and osteocalcin (OC) secretion increased in a dose-dependent manner following dihydroxyvitamin D3 (1,25-D3) treatment when cultured at either temperature. However, AP and 1,25-D3-induced OC levels were elevated in confluent hFOB cells cultured at 39.5°C compared with 33.5°C. Treatment of hFOB cells with 1–34 parathyroid hormone (PTH) resulted in an increase in cAMP levels. Upon reaching confluence, hFOB cultures went through programmed differentiation and formed mineralized nodules as observed by von Kossa staining. Further, immunostaining of postconfluent, differentiated hFOB cells showed that high levels of osteopontin, osteonectin, bone sialoprotein, and type I collagen were expressed. Therefore, the clonal cell line hFOB 1.19 provides a homogeneous, rapidly proliferating model system to study certain stages of human osteoblast differentiation.