Mineralization and the expression of matrix proteins during in vivo bone development.

The in vivo expression of fibronectin, type I collagen, and several non-collagenous proteins was correlated with the development of bone in fetal and early neonatal rat calvariae. Fibronectin was the earliest matrix protein expressed in calvariae, with a peak expression in fetal 16- and 17-day (d) bones. Fibronectin expression coincided with the condensation of preosteoblasts prior to calcification and decreased once bone mineralization commenced. The expression of type I collagen, osteonectin, bone sialoprotein, and alkaline phosphatase mRNAs was found at 17 d. The increase in type I collagen mRNA levels was correlated with a 3.5-fold increase in calcium deposition at 19-20 d. Bone sialoprotein and alkaline phosphatase peaked on fetal 21 d while osteonectin remained at a low level and was localized to the osteoblast layer and the osteocyte lacunae. Osteopontin mRNA levels increased rapidly in neonatal calvariae. After birth, osteonectin and fibronectin were mainly associated with blood vessels. Thus, fibronectin is one of the earliest matrix proteins expressed in calvariae and is rapidly followed by type I collagen, bone sialoprotein, and alkaline phosphatase. Osteocalcin, osteonectin, and osteopontin mRNAs have similar patterns of expression in the developing fetal calvaria, and their synthesis coincided with mineralization.

CD5-mediated specific delivery of DNA to T lymphocytes: compartmentalization augmented by adenovirus.

Specific DNA delivery has been achieved via interactions between an asialoorosomucoid-polylysine conjugate and the asialoglycoprotein receptor. We have now extended this technology to another cell type. In order to achieve DNA delivery uniquely to T cells, we have employed an antibody-polylysine conjugate which binds and is internalized via CD5. Binding analyses of the T101 monoclonal antibody to Jurkat cells and freshly isolated human peripheral T lymphocytes were performed and Scatchard plots revealed Kd values of 1.4 and 1.2 pM, respectively. To introduce DNA into the T cell, a complex of T101-polylysine and the luciferase plasmid was formed (T101-PL-DNA). 125I-labeled antibody alone or T101-PL-DNA complexes were both shown to internalize. Subcellular fractionation indicated that the complex remained in the endosomal compartment of the cell for up to 90 min. However, with the addition of adenovirus particles, there was a decrease of labeled complex in the endosomal fraction over time suggesting it was no longer 'tethered' to the endosome vesicle. In vitro transfections confirmed this result showing the addition of adenovirus particles during incubation resulted in increased expression of the luciferase protein. Without adenovirus, there was limited expression of the transduced gene. These data revealed that T101 can deliver DNA via an antibody-PL conjugate. The addition of adenovirus allowed the DNA to escape the endosome enabling expression of the reporter gene.

The effect of glucocorticoids on osteoblast function. The effect of corticosterone on osteoblast expression of beta 1 integrins.

Prolonged treatment with glucocorticoids is known to produce osteoporosis, which is characterized by a decrease in bone mass. Therefore, we studied the effect of glucocorticoids on the formation of bone and on the expression of beta 1 integrins in a mineralizing organ culture of fetal rat parietal bone. Integrins are a family of integral membrane glycoproteins that mediate the adhesion of cells to extracellular matrix macromolecules and affect the growth and differentiation of cells. In situ hybridization with a 32P-labeled beta 1 integrin cDNA probe was performed on parietal bone, treated with or without 100-nanomolar corticosterone for ninety-six hours, to localize and assess the levels of beta 1 integrin mRNA quantitatively. Corticosterone decreased beta 1 integrin mRNA in the osteoblast layer but not in the periosteum. Northern blot analysis demonstrated a 62 per cent decrease in the levels of beta 1 integrin mRNA in the osteoblast layer of bone that had been stripped of its periosteum. Immunofluorescence microscopy confirmed these results, as they demonstrated a decrease in the levels of beta 1 integrin protein predominantly in the osteoblast layer. This effect was dependent on the concentration of corticosterone. During ninety-six hours of culture, the calcium content and the dry weight of control parietal bone increased 157 per cent and 57 per cent, respectively. However, treatment of these cultures with 100-nanomolar corticosterone inhibited calcification by 24 per cent. The administration of glucocorticoid had no significant effect on the DNA content or dry weight.(ABSTRACT TRUNCATED AT 250 WORDS)

Targeted delivery of DNA using YEE(GalNAcAH)3, a synthetic glycopeptide ligand for the asialoglycoprotein receptor.

In vivo gene therapy shows promise as a treatment for both genetic and acquired disorders. The hepatic asialoglycoprotein receptor (ASGPr) binds asialoorosomucoid-polylysine-DNA (ASOR-PL-DNA) complexes and allows targeted delivery to hepatocytes. The tris(N-acetylgalactosamine aminohexyl glycoside) amide of tyrosyl(glutamyl) glutamate [YEE(GalNAcAH)3] has been previously reported to have subnanomolar affinity for the ASGPr. We have used an iodinated derivative of YEE(GalNAcAH)3 linked to polylysine and complexed to the luciferase gene (pCMV-Luc) in receptor-binding experiments to establish the feasibility of substituting ASOR with the synthetic glycopeptide for gene therapy. Scatchard analyses revealed similar Kd values for both ASOR and the glycopeptide. Binding and internalization of 125I-Suc-YEE(GalNAcAH)3 were competitively inhibited with either unlabeled ASOR or glycopeptide. The reverse was also true; 125I-ASOR binding was competed with unlabeled YEE(GalNAcAH)3 suggesting specific binding to the ASGPr by both compounds. Examination of in vivo delivery revealed that the 125I-labeled glycopeptide complex mimicked previous results observed with 125I-ASOR-PL-DNA. CPM in the liver accounted for 96% of the radioactivity recovered from the five major organs (liver, spleen, kidney, heart, and lungs). Cryoautoradiography displayed iodinated glycopeptide complex bound preferentially to hepatocytes rather than nonparenchymal cells. In vitro, as well as in vivo, transfections using the glycopeptide-polylysine-pCMV-luciferase gene complex (YG3-PL-Luc) resulted in expression of the gene product. These data demonstrate that the YEE(GalNAcAH)3 synthetic glycopeptide can be used as a ligand in targeted delivery of DNA to the liver-specific ASGPr.

Preparation of asialoorosomucoid-polylysine conjugates.

Asialoorosomucoid-polylysine (ASOR-PL) conjugates have been recently developed as carriers of electrostatically bound DNA for targeted delivery to the hepatic asialoglycoprotein receptor (ASGPr) for gene therapy. Using acid-urea gel electrophoresis we have found that previously reported procedures for the fractionation of ASOR-PL conjugates do not efficiently remove noncovalently bound polylysine (PL) from ASOR-PL. DNA complexes prepared with these conjugates have low solubilities, which limits their usefulness for subsequent experimentation, particularly in vivo. For ASOR-PL made by carbodiimide-mediated crosslinking with 5-kDa PL, dialysis against 1 M guanidine hydrochloride is effective to remove the low molecular weight unbound PL. Dialysis is not feasible when using higher molecular weight PLs, but preparative elution acid-urea gel electrophoresis was used to isolate crude ASOR-PL fractions free of unbound PL. ASOR-PL freed of PL by dialysis or electrophoresis was further fractionated by cation-exchange HPLC on carboxymethyl-functionalized columns eluted with a mixed pH-salt gradient. Early-eluting ASOR-PL fractions isolated by a combination of preparative elution acid-urea gel electrophoresis and cation-exchange HPLC were found to be preferred for the formation of soluble DNA complexes.

Synthetic peptide containing Arg-Gly-Asp inhibits bone formation and resorption in a mineralizing organ culture system of fetal rat parietal bones.

The role of integrins, cell surface receptors involved in cell adhesion to the matrix, was studied in a mineralizing organ culture system. Integrin-mediated cell attachment to matrix proteins has been shown to depend partially on the amino acid sequence Arg-Gly-Asp (RGD), present in the extracellular matrix proteins. Therefore, the effect of RGD peptides on bone formation and resorption was studied in the mineralizing organ culture system derived from 18 day fetal rat parietal bones. Addition of 0.1-50 microM GRGDSPK to bones cultured for 4 days inhibited mineralization in a dose-dependent manner as determined by measuring calcium content and % bone/unit area of tissue. A maximal decrease in calcium content and % bone/unit area of 32.5 and 42.9%, respectively, was found with 50 microM GRGDSPK. With 10 and 50 microM GRGDSPK, bone morphology was dramatically altered, with a disruption of osteoblast and mineralized matrix organization. To assess the effect of the peptides on bone resorption, fetal bones were prelabeled in vivo with 45Ca and resorption was stimulated in vitro with parathyroid hormone in the presence or absence of the peptide. A significant decrease in 45Ca release was found with 10 and 50 microM GRGDSPK. Osteoclast number was also significantly decreased on the bone surface. The peptide was not cytotoxic, since no effect on DNA content, dry weight, or collagen synthesis was found. GRADSP, a control peptide, had no significant effect on mineralization, resorption, or other parameters of bone growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucocorticoids inhibit fibronectin synthesis and messenger ribonucleic acid levels in cultured fetal rat parietal bones.

The effects of corticosterone on fibronectin production, bone growth, and morphology were examined in a mineralizing organ culture system derived from 20-day-old fetal rat parietal bones. During 4 days of culture, 1-1000 nM corticosterone had no significant effect on the increase in dry weight or on DNA content, but 100 and 1000 nM corticosterone did inhibit the increase in calcium content. Light microscopic examination of the 4-day cultures demonstrated a glucocorticoid-induced change in osteoblast shape and organization along the mineralizing front of the bone. A dose-dependent inhibition of fibronectin secretion into the medium was determined by enzyme-linked immunosorbent assay. In control cultures, fibronectin production was 0.105 +/- 0.005 microgram/ml.bone at 24 h and 0.397 +/- 0.037 microgram/ml.bone during the 72- to 96-h interval. The maximal inhibition of fibronectin secretion was 45% at 24 h and 70% at 96 h with 1000 nM corticosterone. Both immunofluorescent visualization of fibronectin staining in the tissue and a Western blot of fibronectin in the tissue showed a decrease in fibronectin levels. At 24 and 96 h, a dose- and time-dependent decrease in fibronectin mRNA transcripts was found. At 24 and 96 h, 1000 nM corticosterone produced a decrease of 42% and 62%, respectively, in fibronectin mRNA levels. Our findings show that glucocorticoids inhibit fibronectin production in developing bone. The decrease in fibronectin synthesis may contribute to altered osteoblast organization and function during bone formation.