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S Mohan

Publications and source records attributed to S Mohan.

At least 19 recordsLinked to original sources
PubMed2000

Estrogen receptor specificity in the regulation of skeletal growth and maturation in male mice.

Androgens may regulate the male skeleton directly through a stimulation of androgen receptors or indirectly through aromatization of androgens into estrogen and, thereafter, through stimulation of estrogen receptors (ERs). The relative importance of ER subtypes in the regulation of the male skeleton was studied in ERalpha-knockout (ERKO), ERbeta-knockout (BERKO), and double ERalpha/beta-knockout (DERKO) mice. ERKO and DERKO, but not BERKO, demonstrated decreased longitudinal as well as radial skeletal growth associated with decreased serum levels of insulin-like growth factor I. Therefore, ERalpha, but not ERbeta, mediates important effects of estrogen in the skeleton of male mice during growth and maturation.

Animals↗
PubMed2000

Disproportional skeletal growth and markedly decreased bone mineral content in growth hormone receptor -/- mice.

Growth hormone (GH) is important for skeletal growth as well as for a normal bone metabolism in adults. The skeletal growth and adult bone metabolism was studied in mice with an inactivated growth hormone receptor (GHR) gene. The lengths of femur, tibia, and crown-rump were, as expected, decreased in GHR-/- mice. Unexpectedly, GHR-/- mice displayed disproportional skeletal growth reflected by decreased femur/crown-rump and femur/tibia ratios. GHR-/- mice demonstrated decreased width of the growth plates in the long bones and disturbed ossification of the proximal tibial epiphysis. Furthermore, the area bone mineral density (BMD) as well as the bone mineral content (BMC)/body weight were markedly decreased in GHR-/- mice. The decrease in BMC in GHR-/- mice was not due to decreased trabecular volumetric BMD but to a decreased cross-sectional cortical bone area In conclusion, GHR-/- mice demonstrate disproportional skeletal growth and markedly decreased bone mineral content.

Animals↗
PubMed2000

Development and application of a serum C-telopeptide and osteocalcin assay to measure bone turnover in an ovariectomized rat model.

Biochemical markers applicable to the ovariectomized rat model can provide important tools for studying the bone remodeling process in this animal model of postmenopausal osteoporosis. We describe the development and application of two biochemical markers, a C-telopeptide (of type-I collagen) enzyme-linked immunosorbent assay (ELISA) for measuring bone resorption and an osteocalcin radioimmunoassay (RIA) for measuring bone formation in rat serum. The C-telopeptide ELISA is based on an affinity purified polyclonal antibody generated against human sequence DFSFLPQPPQEKAHDGGR. The antibody epitope involves amino acid sequence, which is similar in rat and human carboxyl terminal peptide of type-I (alpha 1) collagen. Sensitivity of the ELISA was 0.3 ng/ml. The averaged intra- and interassay variation was CV <7%. Averaged dilution and spiked recoveries were 91% and 105%, respectively. The second marker developed is a synthetic peptide-based osteocalcin RIA, which does not require isolation and purification of intact osteocalcin from rat bone. Osteocalcin antiserum used in the RIA was generated in rabbits against a synthetic peptide comprising amino acids 33-49 of the rat osteocalcin sequence. The sensitivity of the RIA was 0.15 ng/ml of peptide. The averaged intra (n = 10) and interassay variations for two controls were CV <9% and 12%, respectively. The averaged dilution and spiked recoveries were 99.6%. In vivo validation of the C-telopeptide ELISA and osteocalcin RIA was performed in an ovariectomized (OVX) rat model. In 12-week-old OVX Sprague Dawley rats, the C-telopeptide and osteocalcin concentrations were approximately 65% and 40%, respectively, higher than the sham group. Estradiol repletion significantly lowered the C-telopeptide and osteocalcin concentration to the levels of the sham group. In addition, changes in serum C-telopeptide concentration correlated negatively with trabecular BMD measured by pQCT (r = -0.51, P < 0.001). In conclusion, the C-telopeptide ELISA and osteocalcin RIA exhibited required sensitivity, accuracy, and adequate discriminatory power to be used for measuring bone resorption and bone formation in the ovariectomized rat model.

Amino Acid Sequence↗
PubMed2000

Insulin-like growth factor system components in hyperparathyroidism and renal osteodystrophy.

BACKGROUND: The insulin-like growth factor (IGF) system plays a key role in regulation of bone formation. In patients with renal osteodystrophy, an elevation of some IGF binding proteins (IGFBPs) has been described, but there is no study measuring serum levels of both IGF-I and IGF-II as well as IGFBP-1 to -6 in different forms of renal osteodystrophy and hyperparathyroidism. METHODS: In a cross-sectional study, we investigated 319 patients with mild (N = 29), moderate (N = 48), preuremic (N = 37), and end-stage renal failure (ESRF; N = 205). The ESRF group was treated by hemodialysis (HD; N = 148), peritoneal dialysis (PD; N = 27), or renal transplantation (RTX; N = 30). As controls without renal failure, we recruited age-matched healthy subjects (N = 87) and patients with primary hyperparathyroidism (pHPT; N = 25). Serum levels of total and free IGF-I, IGF-II, IGFBP-1 to -6, and biochemical bone markers including intact parathyroid hormone (PTH), bone alkaline phosphatase (B-ALP), and osteocalcin (OSC) were measured by specific immunometric assays. IGF system components and bone markers were correlated with clinical and bone histologic findings. Mean values +/- SEM are given. RESULTS: With declining renal function a significant increase was measured for IGFBP-1 (range 7- to 14-fold), IGFBP-2 (3- to 8-fold), IGFBP-3 (1.5- to 3-fold), IGFBP-4 (3- to 19-fold), and IGFBP-6 (8- to 25-fold), whereas IGFBP-5 levels tended to decrease (1.3- to 1. 6-fold). In contrast, serum levels of IGF-I, free IGF-I, and IGF-II remained constant in most patients. Compared with renal failure patients, pHPT patients showed a similar decline in IGFBP-5 levels and less elevated levels of IGFBP-1 (3.5-fold), IGFBP-2 (2-fold), IGFBP-3 (1.2-fold), and IGFBP-6 (4-fold) but no elevation of IGFBP-4 levels. In all subjects, free and total IGF-I levels showed significant negative correlations with IGFBP-1, IGFBP-2, and IGFBP-4 (that is, inhibitory IGF system components) and significant positive correlations with IGFBP-3 and IGFBP-5 (that is, stimulatory IGF system components). A positive correlation was observed between IGF-II and IGFBP-6. ESRF patients with mixed uremic bone disease and histologic evidence for osteopenia revealed significantly (P < 0.05) higher levels of IGFBP-2 and IGFBP-4 but lower IGFBP-5 levels. Histologic parameters of bone formation showed significant positive correlations with serum levels of IGF-I, IGF-II, and IGFBP-5. In contrast, IGFBP-2 and IGFBP-4 correlated positively with indices of bone loss. Moreover, dialysis patients with low bone turnover (N = 24) showed significantly (P < 0.05) lower levels of IGFBP-5, PTH, B-ALP, and OSC than patients with high bone turnover. CONCLUSION: Patients with primary and secondary hyperparathyroidism showed lower levels of the putative stimulatory IGFBP-5 but higher levels of IGFBP-1, -2, -3, and -6, whereas total IGF-I and IGF-II levels were not or only moderately increased. The marked increase in serum levels of IGFBP-4 appeared to be characteristic for chronic renal failure. IGFBP-5 correlated with biochemical markers and histologic indices of bone formation in renal osteodystrophy patients and was not influenced by renal function. Therefore, IGFBP-5 may gain significance as a serological marker for osteopenia and low bone turnover in long-term dialysis patients.

Adult↗
PubMed2000

Diabetic retinopathy is associated with decreased serum levels of free IGF-I and changes of IGF-binding proteins.

In patients with diabetic retinopathy, elevated serum levels of total circulating insulin-like growth factor-I (IGF-I) have been implicated as an important mediator of the disease. There is no study, however, measuring free IGF-I levels in patients with diabetic retinopathy which mediate the biological effects of IGF-I and are modulated by a complex system of six specific IGF binding proteins (IGFBPs) and several IGFBP proteases.

Aged↗
PubMed2000

Evaluation of IGF system component levels and mitogenic activity of uremic serum on normal human osteoblasts.

To test the hypothesis that impairment in bone formation in renal osteodystrophy in adults with chronic renal failure (CRF) might be mediated in part by alterations in circulating levels of the insulin-like growth factor (IGF) system components, we compared serum levels of IGF-I, IGF-II, IGF-binding protein (IGFBP)-3, IGFBP-4 and IGFBP-5 in adults with CRF (CRF patients with parathyroid hormone (PTH) < 100 pg/ml, PTH > 300 pg/ml and end-stage renal failure (ESRF) patients) versus age-matched controls. To evaluate the biological significance of alterations in circulating level of IGF system components, we compared the mitogenic activity of the sera on proliferation of normal human osteoblasts in vitro by using [(3)H]thymidine incorporation. We found severalfold increased serum levels of IGFBP-3 (2-fold), IGFBP-4 (5-fold) and slightly increased IGF-II levels in ESRF patients as well as a 2.6-fold increase in free IGF-I in CRF patients with PTH < 100 pg/ml. The mitogenic activity was found to be increased in serum of kidney failure patients compared to controls. This was most pronounced in CRF patients with PTH < 100 pg/ml showing also a significant increase in free IGF-I and the lowest levels of the IGF-inhibitory IGFBP-4. Our data support the hypothesis that alterations in serum levels of stimulating (i.e. free IGF-I) and inhibitory IGF system components (i.e. IGFBP-4) may influence osteoblastic cell proliferation in renal osteodystrophy.

Alkaline Phosphatase↗
PubMed2000

Studies on human pregnancy-induced insulin-like growth factor (IGF)-binding protein-4 proteases in serum: determination of IGF-II dependency and localization of cleavage site.

Insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4), a consistent inhibitor of IGF action, is subject to proteolytic cleavage by the IGF-II-dependent IGFBP-4 protease. However, regulation of the IGF-II-dependent IGFBP-4 protease in vivo is not known. As IGFBP proteases are known to be triggered during pregnancy, we systematically evaluated the changes in IGFBP-4 proteolysis by serum collected throughout human pregnancy. Results from in vitro protease assays using recombinant IGFBP-4 revealed that IGFBP-4 proteolysis determined in both the presence and absence of exogenous IGF-II significantly increased during the first and second trimesters and reached a plateau by the third trimester. However, in the absence of IGF-II, IGFBP-4 proteolysis by pregnancy serum was only observed after prolonged incubation. IGF-II dose dependently increased IGFBP-4 proteolysis by pregnancy serum, with maximal stimulation observed at a concentration of 0.7 mol/L relative to IGFBP-4. In contrast, IGF-II at an equimolar dose had little effect on proteolysis of recombinant human IGFBP-3, whereas excess IGF-II reproducibly inhibited recombinant human IGFBP-3 proteolysis by pregnancy serum. Although IGF-II enhanced IGFBP-4 proteolysis, results from N-terminal sequence and mass spectrometric analyses of IGFBP-4 proteolytic fragments demonstrate that the cleavage site (Met135-Lys136) in human IGFBP-4 was not altered by IGF-II. Deletion of the residues 121-141 containing this cleavage site blocked IGFBP-4 proteolysis. These findings demonstrate that the increase in IGFBP-4 proteolysis during pregnancy was accounted for mainly by the IGF-II-dependent IGFBP-4 proteolysis. Because IGFBP-4 is a potent inhibitor of IGF actions, it can be speculated that the pregnancy-induced IGFBP-4 proteases may play an important role in regulating fetal growth.

Adult↗
PubMed1999

Progesterone stimulation of human insulin-like growth factor-binding protein-5 gene transcription in human osteoblasts is mediated by a CACCC sequence in the proximal promoter.

Insulin-like growth factor-binding protein-5 (IGFBP-5) is produced by osteoblasts and potentiates insulin-like growth factor mitogenic stimulation in osteoblast cell cultures. Progesterone (PG) increased IGFBP-5 expression in normal human osteoblasts and increased IGFBP-5 transcription in U2 human osteosarcoma cells. We developed a chloramphenicol acetyltransferase reporter construct containing the human IGFBP-5 proximal promoter sequence, which includes TATA and CAAT boxes, and five putative PG response element half-sites. 10(-8) M PG increased promoter activity of this construct in U2 cells co-transfected with a PG receptor isoform A (PR(A)) expression vector. Analysis of 5' deletion constructs indicates that PG transactivation of IGFBP-5 promoter activity does not require the PG response element half-sites but does require the region -162 to -124 containing two tandem CACCC box sequences. Mutation of the proximal CACCC box at -139 eliminated PG transactivation. Gel shift assays using a -162 to -124 DNA fragment, U2 cell nuclear extracts, and purified PR(A) protein indicate that nuclear factors bind to a CACCC sequence at -139 and that PR(A) alters the pattern of transcription factor interaction with the CACCC sequence. Using a luciferase reporter construct containing base pairs -252 to +24 of the IGFBP-5 promoter, we found that both PR(A) and PR(B) isoforms mediated PG stimulation of promoter activity. These results suggest that PG may stimulate IGFBP-5 gene transcription via a novel mechanism involving PR and CACCC-binding factors.

Base Sequence↗
PubMed1999

The insulin-like growth factor axis and prostate cancer: lessons from the transgenic adenocarcinoma of mouse prostate (TRAMP) model.

We have characterized the temporal expression of the insulin-like growth factor (IGF) axis in the transgenic adenocarcinoma of mouse prostate (TRAMP) model as prostate cancer progression in this model closely mimics that observed in the human disease, and the model provides samples representing the earliest stages of prostate cancer that are clinically the most difficult to obtain. We report that prostate-specific IGF-I mRNA expression increased during prostate cancer progression in TRAMP mice and was elevated in the accompanying metastatic lesions, whereas prostatic IGF-I mRNA remained at nontransgenic levels in androgen-independent disease. Expression of IGF-II mRNA, however, was reduced in primary prostate cancer, metastatic lesions, and androgen-independent disease. Expression of type-1 IGF receptor (IGF1R) mRNA, encoding the cognate receptor for both IGF-I and IGF-II, as well as type-2 IGF receptor (IGF2R) mRNA was not found to be altered during primary prostate cancer progression in intact TRAMP mice but was dramatically reduced in metastatic lesions and in androgen-independent disease. Similar to reports from clinical disease, serum IGF-I levels were observed to increase precociously in TRAMP mice early in disease progression but remained at nontransgenic levels after castration. Elevated serum levels of IGF-binding protein 2 were observed to correlate with advanced prostate cancer in the TRAMP model. Together these observations implicate IGF-I as an important factor during the initiation and progression of primary prostate cancer and provide evidence that there is a strong selection against expression of IGF1R and IGF2R in metastatic and androgen-independent disease.

Adenocarcinoma↗
PubMed1999

Structural differences among monoclonal antibodies with distinct fine specificities and kinetic properties.

The mAbs HyHEL-8, HyHEL-26 (HH8, and HH26, respectively) recognize epitopes on hen egg-white lysozyme (HEL) highly overlapping with the structurally defined HH10 epitope, while the structurally related XRPC-25 is specific for DNP and does not bind HEL. All four Abs appear to use the same Vk23 germ line gene, and all but HH8 use the same VH36-60 germ line gene. Of the three anti-HEL Abs, the sequences of HH26 variable regions are closest to those encoded by the respective germ line sequences. HH8 utilizes a different member of the VH36-60 gene family. Thus, the same Vk and VH genes, combined with somatically derived sequence differences, are used to recognize the unrelated Ags HEL and DNP. In contrast, different VH36-60 germ line genes are used to bind the same antigen (e.g. HH8 vs HH10 and HH26). While the affinities of HH10, HH8, and HH26 for HEL vary by less than 10-fold, their affinities for mutated Ag vary over several orders of magnitude. Analyses of Fab binding kinetics with natural species variants and site-directed mutants of lysozyme indicate that these cross-reactivity differences reflect the relative sensitivities of both the association and dissociation rates to antigenic mutation: HH8 has relatively mutation-insensitive association and dissociation rates, HH10 has a relatively mutation-sensitive association rate but more variable dissociation rates, and HH26 has variable association and dissociation rates. Only a few amino acid differences among the antibodies produce the observed differences in the robustness of the association and dissociation rates. Our results suggest that association and dissociation rates and mutation sensitivity of these rates may be independently modulated during antibody repertoire development.

Animals↗
PubMed1999

The diagnosis and treatment of osteoporosis: future prospects.

Osteoporosis is a common disease that affects millions of patients throughout the world. We anticipate that both the diagnosis and the treatment of this disease will be revolutionized by the integration of genomics and informatics. It is predicted that a genetic algorithm will be developed to identify at-risk patients before they develop osteoporosis, so that preventive measures can be instituted. The sequencing of the human genome will lead to revolutionary advances in at least three areas of osteoporosis therapy: small molecule therapy, protein therapy and gene therapy. One area of focus for future therapeutics in osteoporosis will be on osteogenic agents, which should have a high likelihood of success because the skeleton has the innate capacity to regenerate itself.

Animals↗
PubMed1999

Heterogeneous spectrum of mutations in the Fanconi anaemia group A gene.

Fanconi anaemia (FA) is a genetically heterogeneous autosomal recessive disorder associated with chromosomal fragility, bone-marrow failure, congenital abnormalities and cancer. The gene for complementation group A (FAA), which accounts for 60-65% of all cases, has been cloned, and is composed of an open reading frame of 4.3 kb, which is distributed among 43 exons. We have investigated the molecular pathology of FA by screening the FAA gene for mutations in a panel of 90 patients identified by the European FA research group, EUFAR. A highly heterogeneous spectrum of mutations was identified, with 31 different mutations being detected in 34 patients. The mutations were scattered throughout the gene, and most are likely to result in the absence of the FAA protein. A surprisingly high frequency of intragenic deletions was detected, which removed between 1 and 30 exons from the gene. Most microdeletions and insertions occurred at homopolymeric tracts or direct repeats within the coding sequence. These features have not been observed in the other FA gene which has been cloned to date (FAC) and may be indicative of a higher mutation rate in FAA. This would explain why FA group A is much more common than the other complementation groups. The heterogeneity of the mutation spectrum and the frequency of intragenic deletions present a considerable challenge for the molecular diagnosis of FA. A scan of the entire coding sequence of the FAA gene may be required to detect the causative mutations, and scanning protocols will have to include methods which will detect the deletions in compound heterozygotes.

Base Sequence↗
PubMed1999

Normal growth despite GH, IGF-I and IGF-II deficiency.

A 6.5-year-old male with normal linear growth, despite septo-optic dysplasia, panhypopituitarism and a deficient GH/IGF axis, is presented. In addition to measuring IGF-I, IGF-II and IGFBP-3, serum IGFBP-1, -2, -4 and -5 were measured. A human osteosarcoma cell line was used to assess growth-promoting activity in the patient's serum. The role of leptin in linear growth in this case was investigated. There was no evidence for hyperinsulinism or hyperandrogenism. GH was undetectable upon multiple stimulation. GHBP was elevated. Serum IGF-I (25 microg/l), IGF-II (194 microg/l), IGFBP-3 (0.4 mg/l), and IGFBP-5 (87 microg/l) levels were low compared to age-matched prepubertal children. Serum IGFBP-4 level was normal. Molecular size of IGF-II in the patient's serum was normal, suggesting normal IGF-II bioavailability. Human osteosarcoma cell proliferation in response to the patient's serum was similar to sera from age-matched normal controls. Leptin levels were markedly elevated. Osteoblast cell proliferation was not stimulated by leptin. The data demonstrate that normal growth and osteoblast cell proliferation in this patient is not mediated by GH, total IGFs, insulin, or leptin, and suggest the presence of a yet unidentified growth factor or mechanism. The case offers a detailed picture of binding proteins in a case of growth without GH. It introduces osteoblast cell proliferation as a method of assessing serum growth-promoting activity in such cases. It adds IGF-II and leptin to the list of excluded growth-promoting candidates in GH-independent growth, and further demonstrates our incomplete understanding of the phenomenon of growth.

Body Height↗
PubMed1999

Serum levels of insulin-like growth factor binding proteins in Ecuadorean children with growth hormone insensitivity.

Although insulin-like growth factor binding proteins (IGFBPs) are known to be important modulators of the action of insulin-like growth factors (IGFs), regulation of their production in vivo is not completely understood. Serum concentrations of IGFBP-3, -4 and -5 and acid-labile subunit (ALS) were therefore examined in 20 children with growth hormone (GH) insensitivity before and after 6 months of therapy with recombinant human IGF-I (80 or 120 micrograms/kg twice daily). The IGFBP concentrations in these children were compared with those in 62 GH-deficient children receiving GH therapy for 3 months. Serum levels of IGFBP-3, -4 and -5 and ALS all increased significantly (p < 0.0001) in GH-deficient children in response to GH therapy, whereas no significant increases occurred in the children with GH insensitivity. These findings indicate that GH is responsible for the regulation of serum levels of IGFBP-3, -4 and -5 and ALS, and that IGF-I does not directly regulate the concentrations of these circulating IGFBPs.

Body Height↗
PubMed1999

Regulation of low shear flow-induced HAEC VCAM-1 expression and monocyte adhesion.

We recently reported that prolonged exposure of human aortic endothelial cells (HAEC) to low shear stress flow patterns is associated with a sustained increase in the activated form of the transcriptional regulator nuclear factor-kappaB (NF-kappaB). Here we investigate the hypothesis that low shear-induced activation of NF-kappaB is responsible for enhanced expression of vascular cell adhesion molecule (VCAM-1) resulting in augmented endothelial cell-monocyte (EC-Mn) adhesion and that this activation is dependent on intracellular oxidant activity. Before exposure to low shear (2 dyn/cm2) for 6 h, HAEC were preincubated with or without the antioxidants pyrrolidine dithiocarbamate (PDTC) or N-acetyl-L-cysteine (NAC). PDTC strongly inhibited low shear-induced activation of NF-kappaB, expression of VCAM-1, and EC-Mn adhesion. Paradoxically, NAC exerted a positive effect on low shear-induced VCAM-1 expression and EC-Mn adhesion and only slightly downregulated NF-kappaB activation. However, cytokine-induced NF-kappaB activation and VCAM-1 expression are blocked by both PDTC and NAC. These data suggest that NF-kappaB plays a key role in low shear-induced VCAM-1 expression and that pathways mediating low shear- and cytokine-induced EC-Mn adhesion may be differentially regulated.

Acetylcysteine↗
PubMed1999

Antigen trafficking and accessory cell function in respiratory epithelial cells.

We investigated accessory cell function, antigen (Ag) trafficking, and uptake of immune complexes in isolated nasal epithelial cells (NEC) and airway epithelial cells (AEC), as well as in the two respiratory epithelial cell lines A549 and BEAS-2B. The NEC and AEC were capable of supporting Ag-specific as well as phytohemagglutinin-induced and anti-CD3 antibody-induced T-cell proliferation. We colocalized fluorescein isothiocyanate (FITC)-labeled Ags with human leukocyte antigen (HLA)-DR in A549 and BEAS-2B, utilizing laser confocal microscopy. Respiratory epithelial cells stimulated and unstimulated with interferon (IFN)-gamma were pulsed with FITC-labeled Ags for varying periods and evaluated for their ability to internalize Ag. In the unstimulated cells, intracellular punctate staining was evident at 60 min and persisted up to 120 min. In the IFN-gamma-stimulated cells (100 U/ml for 48 h), uptake occurred at 30 min, was maximal at 60 min, and diminished at 120 min. We conducted kinetic studies in the A549 and BEAS-2B cells, utilizing electron microscopy with colloidal gold-conjugated Ags (Au-OVA). At 15 min, Au-OVA was evident in the early compartments resembling the compartment of uncoupling of receptor and ligand. At 30 min, multivesicular bodies were labeled with Au-OVA, and by 60 min Au-OVA was present in the primary and secondary lysosomes. The FITC-labeled Ags colocalized with an early endosomal marker (anti-cathepsin D), a late endosomal marker (M6PR), a lysosomal marker (CD63), and with 3-(2, 4-dinitroanilino)-3'-aminomethyldipropylamine, a marker of acidic vesicles. The BEAS-2B and A549 cells, and NEC and AEC, expressed surface Fcgamma receptor and internalized IgG immune complexes. The NEC and AEC also expressed the costimulatory molecules CD80 and CD86 as determined with flow cytometry, the reverse transcription-polymerase chain reaction for RNA, and immunohistochemistry, and T-cell proliferation could be blocked by treating NEC and AEC with anti-CD80 and anti-CD86 antibodies. Our findings suggest that respiratory epithelial cells may have a role in local Ag presentation.

Antigen-Antibody Complex↗
PubMed1999

Recombinant human insulin-like growth factor-binding protein-5 stimulates bone formation parameters in vitro and in vivo.

Insulin-like growth factor-binding protein-5 (rhIGFBP-5) is stored in bone and stimulates osteoblast cell proliferation in vitro. Bone formation is dependent on the number and activity of osteoblasts. We therefore evaluated the ability of recombinant human (rh) IGFBP-5 to increase osteoblast activity in vitro; both alkaline phosphatase (ALP) activity and osteocalcin levels showed a dose-dependent increase. In in vivo time-course studies, daily s.c. administration of 50 microg rhIGFBP-5/day/mouse significantly increased serum osteocalcin levels by day 7, and these levels were sustained through day 21. We further evaluated whether rhIGFBP-5 was as effective as IGF-I. Daily s.c. administration of rhIGFBP-5 (50 microg/day), IGF-I (13 microg/day), or IGF-I plus rhIGFBP-5 complex for 9 days increased serum osteocalcin levels by 58%, 65%, and 81% (P < 0.001 in all) and femoral bone extract ALP activity by 85% (P < 0.001), 29% (P < 0.05), and 13% (P = NS), respectively, and decreased carboxyl-terminal cross-linked telopeptide of type I collagen by 29% (P < 0.05), 20% (P = NS), and 12.5% (P = NS), respectively. One s.c. injection of rhIGFBP-5 (50 microg/mouse) increased serum osteocalcin and bone ALP activity by 21% (P < 0.05) and 27% (P < 0.02), respectively, after 5 days, but did not significantly increase serum IGF-I (1, 6, or 24 h/postinjection), suggesting that the effects of rhIGFBP-5 on bone are not mediated by increasing circulating IGF-I. Our data demonstrate that systemic administration of rhIGFBP-5, either alone or in combination with IGF-I, increases bone formation parameters in vivo.

Alkaline Phosphatase↗