The selective vitamin D receptor activator for albuminuria lowering (VITAL) study: study design and baseline characteristics.

BACKGROUND: Patients with diabetic nephropathy are at high risk for further progressive renal function loss. Treatments that decrease albuminuria have been linked with renal and cardiovascular protection. However, even when taking optimal treatment, residual renal and cardiovascular risk remains high which correlates with the magnitude of residual albuminuria. Use of vitamin D receptor activators, such as calcitriol and paricalcitol, is associated with improved sur- vival. A small study with paricalcitol showed reductions in albuminuria. The VITAL study tests the hypothesis whether paricalcitol persistently reduces albuminuria in diabetic subjects already receiving angiotensin-converting enzyme inhibitor (ACEI) and/or angiotensin receptor blocker (ARB) therapy. METHODS: Randomization in this double-blind trial is equal allocation to paricalcitol 1 micro/day, 2 microg/day, or placebo. Inclusion criteria include: a diagnosis of type 2 diabetes, urinary albumin/creatinine ratio (UACR) between 100-3,000 mg/g, estimated glomerular filtration rate (eGFR) between 15-90 ml/min/1.73 m(2), serum calcium <9.8 mg/dl, and parathyroid hormone (PTH) between 35-500 pg/ml. RESULTS: Baseline characteristics of the 281 subjects are: 69% men, mean age 64.9 +/- 10.4 years, eGFR 40.7 +/- 16.7 ml/min, median UACR (interquartile range) 612.3 mg/g (281-1,181 mg/g) and PTH 98.4 +/- 63.8 pg/ml. CONCLUSION: This trial will be the first clinical test of the hypothesis that paricalcitol possesses pleiotropic effects and can modulate albuminuria in the setting of ACEI and/or ARB therapy. Results will have important clinical implications and are expected in November 2009.

Adynamic bone in patients with chronic kidney disease.

Adynamic bone in patients with chronic kidney disease (CKD) is a clinical concern because of its potential increased risk for fracture and cardiovascular disease (CVD). Prevalence rates for adynamic bone are reportedly increased, although the variance for its prevalence and incidence is large. Differences in its prevalence are largely attributed to classification and population differences, the latter of which constitutes divergent groups of elderly patients having diabetes and other comorbidities that are prone to low bone formation. Most patients have vitamin D deficiency and the active form, 1,25-dihydroxyvitamin D, invariably decreases to very low levels during CKD progression. Fortunately, therapy with vitamin D receptor activators (VDRAs) appears to be useful in preventing bone loss, in part, by its effect to stimulate bone formation and in decreasing CVD morbidity, and should be considered as essential therapy regardless of bone turnover status. Future studies will depend on assessing cardiovascular outcomes to determine whether the risk/reward profile for complications related to VDRA and CKD is tolerable.

Prevalence and severity of disordered mineral metabolism in Blacks with chronic kidney disease.

Disorders of mineral metabolism develop early in chronic kidney disease, but it appears that Blacks with stage-5 disease have more severe secondary hyperparathyroidism than other races. We measured levels of parathyroid hormone, calcium, phosphorus, 25-hydroxyvitamin D (25D) and 1,25-dihydroxyvitamin D (1,25D) in 227 Black and 1633 non-Black participants in the SEEK study, a multi-center cohort of patients with early chronic kidney disease. Overall, Blacks had similar 1,25D levels compared with non-Blacks, but significantly lower levels of 25D with higher levels of calcium, phosphorus, and parathyroid hormone, and were significantly more likely to have hyperphosphatemia than non-Blacks. In multivariable analyses adjusted for age, gender, estimated glomerular filtration rate, body mass index, and diabetes, Blacks had significantly lower 25D and higher parathyroid hormone levels than non-Blacks, with the latter parameter remaining significant after further adjustment for calcium, phosphorus, 25D, and 1,25D. The association between Black race and secondary hyperparathyroidism, independent of known risk factors, suggests that novel mechanisms contribute to secondary hyperparathyroidism in Blacks with chronic kidney disease.

Prevalence of abnormal serum vitamin D, PTH, calcium, and phosphorus in patients with chronic kidney disease: results of the study to evaluate early kidney disease.

Abnormalities of mineral metabolism occur early in chronic kidney disease. Quantification of the prevalence of these abnormalities has not been described using current assays nor in large unselected populations. This outpatient cohort cross-sectional study was conducted in 153 centers, (71% primary care practices). Blood for parathyroid hormone (PTH), vitamin D metabolites, creatinine, calcium (Ca), and phosphorus (P) were drawn between June and October 2004. Low 1,25-dihydroxyvitamin D (1,25 OH2 D3) was defined as <22 pg/ml. The 1814 patients had a mean age of 71.1 years old; 48% had diabetes mellitus (DM). Low 1,25 OH2 D3 was evident at all estimated glomerular filtration rate (eGFR) levels: 13% in those with eGFR >80 ml/min, >60% in those with eGFR <30 ml/min. High PTH (>65pm/dl) occurred in 12% with eGFR >80 ml/min. Serum Ca and P were normal until eGFR was <40 ml/min. Significant differences in the mean and median values of 1,25 OH2 D3, PTH, but not 25(OH)D3 levels, were seen across deciles of eGFR (P<0.001). Multivariate analysis revealed that DM, increased urinary albumin/creatinine ratio and lower eGFR predicted lower values of 1,25 OH2 D3. A high prevalence of mineral metabolite abnormalities occurs in a large unreferred US cohort. Low 1,25 OH2 D3 and elevated PTH are common at higher eGFR than previously described. As bone, cardiovascular disease, and mineral metabolite are correlated; further studies are necessary to determine the importance of these findings relative to outcomes.

Vitamin D in chronic kidney disease: a systemic role for selective vitamin D receptor activation.

Hyperparathyroidism occurs in most patients during the progression of chronic kidney disease (CKD) and one of its initiating events, reduced serum levels of 1,25-dihydroxyvitamin D, results from a decrease in renal 1alpha hydroxylase activity, which converts 25-hydroxyvitamin D to its activated form. The combination of persistently high parathyroid hormone (PTH) and low 1,25-dihydroxyvitamin D is associated with bone loss, cardiovascular disease, immune suppression and increased mortality in patients with end-stage kidney failure. Recent studies in dialysis patients suggest that paricalcitol, a selective activator of the vitamin D receptor (VDR), is associated with a more favorable efficacy to side effect profile than calcitriol, with less morbidity and better survival. One hypothesis derived from such studies suggests that systemic activation of VDRs may have direct effects on the cardiovascular system to decrease mortality in CKD. Although current guidelines for regulating serum calcium, phosphate and PTH recommend specific interventions at the various stages of CKD to prevent or postpone irreversible parathyroid disease and decrease cardiovascular morbidity and mortality, emerging data suggest that vitamin D therapy may prolong survival in this patient population by mechanisms that are independent of calcium, phosphate and PTH. It is suggested that a re-evaluation of current treatment recommendations is needed and that future research should focus on mechanisms that distinguish potential tissue specific benefits of selective VDR activators in patients with CKD.

Cytokine accumulation in osteitis fibrosa of renal osteodystrophy

Bone marrow fibrosis occurs in association with a number of pathological states. Despite the extensive fibrosis that sometimes characterizes renal osteodystrophy, little is known about the factors that contribute to marrow accumulation of fibrous tissue. Because circulating cytokines are elevated in uremia, possibly in response to elevated parathyroid hormone levels, we have examined bone biopsies from 21 patients with end-stage renal disease and secondary hyperparathyroidism. Bone sections were stained with antibodies to human interleukin-1alpha (IL-1alpha), IL-6, IL-11, tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-ß (TGF-ß) using an undecalcified plastic embedding method. Intense staining for IL-1alpha, IL-6, TNF-alpha and TGF-ß was evident within the fibrotic tissue of the bone marrow while minimal IL-11 was detected. The extent of cytokine deposition corresponded to the severity of fibrosis, suggesting their possible involvement in the local regulation of the fibrotic response. Because immunoreactive TGF-ß and IL-6 were also detected in osteoblasts and osteocytes, we conclude that selective cytokine accumulation may have a role in modulating bone and marrow cell function in parathyroid-mediated uremic bone disease

Cytokine accumulation in osteitis fibrosa of renal osteodystrophy.

Bone marrow fibrosis occurs in association with a number of pathological states. Despite the extensive fibrosis that sometimes characterizes renal osteodystrophy, little is known about the factors that contribute to marrow accumulation of fibrous tissue. Because circulating cytokines are elevated in uremia, possibly in response to elevated parathyroid hormone levels, we have examined bone biopsies from 21 patients with end-stage renal disease and secondary hyperparathyroidism. Bone sections were stained with antibodies to human interleukin-1alpha (IL-1alpha), IL-6, IL-11, tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) using an undecalcified plastic embedding method. Intense staining for IL-1alpha, IL-6, TNF-alpha and TGF-beta was evident within the fibrotic tissue of the bone marrow while minimal IL-11 was detected. The extent of cytokine deposition corresponded to the severity of fibrosis, suggesting their possible involvement in the local regulation of the fibrotic response. Because immunoreactive TGF-beta and IL-6 were also detected in osteoblasts and osteocytes, we conclude that selective cytokine accumulation may have a role in modulating bone and marrow cell function in parathyroid-mediated uremic bone disease.

Intravenous versus oral vitamin d therapy in dialysis patients: what is the question?

The debate regarding the administration of vitamin D (parenteral versus pulse oral) in dialysis patients has centered on the efficacy of parathyroid hormone (PTH) suppression while ignoring other questions related to complications and compliance. Past studies looking at efficacy showed no differences during short-term treatment, although the small number of patients studied reduces the significance of these findings. Long-term studies with larger populations have shown that parenteral calcitriol is more effective than pulse oral calcitriol in suppressing PTH. When considering the questions of complications and compliance the current literature demonstrates that parenteral vitamin D therapy is associated with fewer episodes of hypercalcemia and hyperphosphatemia and that patients receiving pulse oral calcitriol require more phosphate binders. Because of the documented high noncompliance rate with oral medications in the dialysis population, parenterally administered vitamin D is expected to more completely suppress PTH long term and result in fewer parathyroidectomies. Based on these considerations it is suggested that parenteral vitamin D analogs are superior to pulse oral calcitriol for the long-term control of hyperparathyroidism in dialysis patients.

IGF-binding protein-5 stimulates osteoblast activity and bone accretion in ovariectomized mice.

Insulin-like growth factor binding protein-5 (IGFBP-5) is an osteoblast secretory protein that becomes incorporated into the mineralized bone matrix. In osteoblast cultures, IGFBP-5 stimulates cell proliferation by an IGF-independent mechanism. To evaluate whether IGFBP-5 can stimulate osteoblast activity and enhance bone accretion in a mouse model of osteoblast insufficiency, daily subcutaneous injections of either intact [IGFBP-5 (intact)] or carboxy-truncated IGFBP-5 [IGFBP-5-(1--169)] were given to ovariectomized (OVX) mice for 8 wk. Femur and spine bone mineral density (BMD), measured every 2 wk, showed early and sustained increases in response to IGFBP-5. Bone histomorphometry of cancellous bone showed significant elevations in the bone formation rate in both the femur metaphysis [IGFBP-5- (1)] only) and spine compared with OVX controls. IGFBP-5 also stimulated osteoblast number in the femur IGFBP-5-(1--169) only) and spine. These data indicate that IGFBP-5 effectively enhances bone formation and bone accretion in OVX mice by stimulating osteoblast activity. The finding that IGFBP-5-(1--169) is bioactive in vivo indicates that the carboxy-terminal portion is not required for this bone anabolic effect.

Vitamin D analogs in uremia: integrating medical and nutritional issues.

A system of regulatory molecules interacts at the cellular level to control and coordinate the many metabolic pathways that constitute normal mineral metabolism. Alterations that occur in uremia profoundly disrupt this intricate system of regulation. A lack of control poses serious consequences for patients with chronic renal disease, and restoring some level of control represents a significant treatment goal. To achieve adequate treatment, it is necessary to correct aberrations in the metabolism of the major regulatory molecules, parathyroid hormone, vitamin D, calcium, and phosphorus. The use of vitamin D hormone replacement therapy is one important part of this strategy, and the availability of newer vitamin D compounds may prove to be especially beneficial. The effective use of these compounds, nevertheless, depends on the coordinated efforts of each member of the health care team to design and implement an integrated treatment protocol that recognizes all aspects of intervention.

IGFBP-5(201-218) stimulates Cdc42GAP aggregation and filopodia formationin migrating mesangial cells.

BACKGROUND: We have previously shown that insulin-like growth factor-I (IGF-I) and IGF binding protein-5 (IGFBP-5) induce mesangial cell migration using separate stimulatory and effector pathways. The IGFBP-5 stimulatory pathway is mediated by the serine/threonine kinase IGFBP-5 receptor, which is activated by the carboxy-terminal peptide IGFBP-5201-218. In this study, we examined the direct effects of IGFBP-5201-218 on stimulatory and effector pathways that lead to a change in mesangial cell (MC) phenotype. METHODS: Rapid actin reorganization, formation of filopodia, and characterization of novel substratum attachment structures that develop during IGFBP-5-mediating migration were examined by light, immunofluorescence, and electron microscopy. Using a wounding assay, migration was measured after the addition of stimulants and inhibitors. RESULTS: Stimulation of MCs with IGFBP-5201-218 induces rapid actin reorganization and loss of peripheral focal adhesions. The MCs develop long cellular extensions where f-actin and beta-actin terminate in unique substratum attachments. Fluorescence microscopy of stimulated cells shows that Cdc42GAP aggregates within minutes following treatment with IGFBP-5201-218. In contrast, IGF-I increases staining for Rac-1, but not Cdc42GAP, in association with the formation of prominent leading lamellae without filopodia. Staurosporin inhibits cell migration and Cdc42GAP aggregation only when added within the first hour, suggesting that it inhibits the stimulatory effect of IGFBP-5201-218 by blocking the IGFBP-5 receptor serine/threonine kinase activity. CONCLUSIONS: These data demonstrate that IGFBP-5201-218 preferentially activates Cdc42 and induces the formation of long filopodia with unique substratum attachments that produce a novel mode of locomotion.

Insulin-like growth factor binding protein-5 proteolytic activity in ovine articular chondrocyte culture.

We have previously demonstrated that ovine articular chondrocytes synthesise and release insulin-like growth factor binding protein-5 (IGFBP-5) which subsequently undergoes proteolysis in the tissue culture medium. The IGFBP-5 proteolytic activity has now been characterised and its substrate specificity analysed using recombinant IGFBP-5 and purified chondrocyte-derived IGFBPs. Iodinated human recombinant IGFBP-5 was incubated with chondrocyte culture or conditioned medium in the presence or absence of various inhibitors. Serine protease inhibitors aprotinin and heparin effectively inhibited the breakdown of IGFBP-5. Furthermore, insulin-like growth factor-I (IGF-I) but not its structural analogues with reduced affinity for IGFBP-5, was also able to partially protect IGFBP-5 from degradation indicating that the association of IGF with the binding protein was required for the inhibition of the proteolytic activity. The inflammatory cytokine interleukin-1 did not have any effect on IGFBP-5 proteolysis. The proteolytic activity appears to be IGFBP-5-specific since the incubation of chondrocyte-derived IGFBPs with chondrocyte conditioned medium resulted in the loss of IGFBP-5 while the levels of the other two IGFBPs (IGFBP-2 and a 24 kDa IGFBP) remained unchanged. In conclusion, we show that IGFBP-5 is specifically cleaved by a serine protease released by primary cultures of ovine articular chondrocytes and also demonstrate the ability of IGF-I to inhibit the proteolytic activity both in cell culture and in cell-free conditions.

Regulation of insulin-like growth factor-binding protein-5 by insulin-like growth factor I and interleukin-1alpha in ovine articular chondrocytes.

Insulin-like growth factors (IGFs) contribute to the maintenance of the cartilage matrix by stimulating proteoglycan synthesis. In contrast, interleukin-1 (IL-1), an inflammatory cytokine, suppresses the synthesis of proteoglycans. In pathological conditions the chondrocytes' responsiveness to IGF-I is decreased, and elevated levels of IGF-binding proteins (IGFBPs) have been implicated as a possible cause. The aim of this study was to investigate the effects of IGF-I and IL-1 on IGFBP production by ovine articular chondrocytes (OAC) and the roles of these IGFBPs in the regulation of proteoglycan synthesis. As revealed by Western ligand and immunoblotting, OACs secreted IGFBP-2 and a 24-kDa IGFBP in culture medium under basal conditions. Exposure of the cells to IGF-I for 48 h resulted in the appearance of IGFBP-5 in the medium. Des(1-3)IGF-I, an IGF-I analog with reduced affinity for IGFBPs, also increased the level of IGFBP-5, but to a lesser extent than IGF-I, whereas LR3IGF-I, which has virtually no affinity for IGFBPs, had no effect on IGFBP-5. Furthermore, IGFBP-5 underwent a time-dependent limited proteolysis when incubated with OAC-conditioned medium, degrading into 22- and 16-kDa fragments. The degradation of IGFBP-5 was significantly inhibited by IGF-I, but not by des(1-3)IGF-I or LR3IGF-I. Basic fibroblast growth factor, transforming growth factor-beta, and platelet-derived growth factor had no effect on OAC IGFBPs. However, IL-1alpha increased the IGFBP-5 level in a dose-dependent manner, showing maximum activity at 200 U/ml. Furthermore, IL-1alpha, but not IGF-I, induced IGFBP-5 messenger RNA expression, as assessed by Northern blot analysis. Coincubation of IGF-I with IL-1alpha resulted in a substantially increased IGFBP-5 protein level, suggesting a synergism between the mechanisms of action of these two factors. Des(1-3)IGF-I and LR3IGF-I were 10 times more potent than IGF-I in stimulating proteoglycan synthesis, indicating inhibition of IGF-I activity by endogenous IGFBPs. IL-1alpha reduced the IGF-I bioactivity, but had no effect on the activities of the IGF-I analogs, thus implying that locally produced IGFBPs, particularly IGFBP-5, which was substantially increased when IGF-I and IL-1alpha were coincubated, mediated the reduction of the IGF-I activity. Our results demonstrate that IGF-I and IL-1alpha synergistically increase the level of IGFBP-5 in OAC by inhibiting the proteolysis and stimulating the expression of IGFBP-5, respectively. Furthermore, the attenuation of IGF-I-stimulated proteoglycan synthesis by IL-1alpha in OAC appears to be mediated by chondrocyte IGFBPs. We conclude that locally produced IGFBPs, in particular IGFBP-5, may play a critical role in the regulation of cartilage matrix degradation in inflammatory and degenerative arthritides.

Insulin-like growth factor-binding protein-5 (IGFBP-5) stimulates phosphorylation of the IGFBP-5 receptor.

The finding that insulin-like growth factor (IGF)-binding protein-5 (IGFBP-5) binding to mouse osteoblasts was capable of being downregulated by IGFBP-5 suggested that the 420-kDa membrane protein, which interacted with IGFBP-5, may be a signaling receptor (Andress, D. L. J. Biol. Chem. 270: 28289-28296, 1995). In the current study, a carboxy-terminal IGFBP-5 peptide, IGFBP-5-(201-218), which was found to competitively inhibit 125I-IGFBP-5 binding and to specifically bind to osteoblast monolayers, was used to affinity-purify the 420-kDa membrane protein. Co-incubation of the affinity-purified membrane protein with [32P]ATP resulted in autophosphorylation at serine residues. Serine phosphorylation of the 420-kDa protein was enhanced by intact IGFBP-5, IGFBP-5-(1-169), and IGFBP-5-(201-218). When the IGFBP-5 receptor was incubated with dephosphorylated casein in the presence of [32P]ATP, casein became phosphorylated on serine residues. These data indicate that IGFBP-5 stimulates the phosphorylation of the IGFBP-5 receptor and suggest that serine/threonine kinase activation may be important in mediating some of the IGF-independent effects of IGFBP-5.

A calcimimetic agent acutely suppresses parathyroid hormone levels in patients with chronic renal failure. Rapid communication.

The control of hyperparathyroidism in patients with chronic renal failure continues to be a problem, particularly when parathyroid hormone (PTH) suppression becomes refractory to calcitriol activation of parathyroid cell 1,25-dihydroxyvitamin D receptors. To evaluate whether parathyroid cell calcium receptor activation may be useful in suppressing PTH levels, we tested the safety and effectiveness of a novel calcimimetic agent in dialysis patients with hyperparathyroidism. In a prospective, dose finding study, the calcimimetic agent, NPS R-568, was administered orally to seven patients at the start of a hemodialysis session and again 24 hours later. Plasma PTH, calcitonin and ionized calcium levels were measured over a 48 hour period and patients were observed for adverse events. Plasma PTH levels fell abruptly in all patients after a single dose of the compound, with the maximum suppression occurring within one to two hours after its administration. Following the administration of low doses (40 or 80 mg), the suppressed PTH levels rose to baseline values over 48 hours, whereas in patients who received high doses (120 or 200 mg) the mean PTH level remained 51% below baseline. Plasma calcitonin increased after the administration of both low and high doses (peak effect within 4 to 6 hr), with levels always returning to baseline by 48 hours. There were no episodes of hypocalcemia and no adverse effects were reported. We conclude that the activation of parathyroid cell calcium receptors by a novel calcimimetic compound is safe and effective in acutely suppressing PTH secretion in dialysis patients with hyperparathyroidism. Whether concomitant stimulation of calcitonin secretion will provide added beneficial effects on bone remodeling remains to be determined in long-term studies.

Evidence that serum phosphate is independently associated with serum PTH in patients with chronic renal failure.

There has been controversy regarding the initial pathogenic events involved with the hyperparathyroidism of chronic renal failure (CRF). Low serum levels of 1,25-dihydroxyvitamin D in uremic patients are postulated by some as having a role in permitting higher parathyroid hormone (PTH) secretion. However, recent animal and in vitro studies strongly suggest that phosphate has a direct effect on parathyroid cells to enhance PTH secretion. To evaluate the relationships among serum phosphate, calcium, PTH, and 1,25-dihydroxyvitamin D in uremic humans, we performed a cross-sectional analysis of 84 patients with varying levels of CRF. Using stepwise regression analysis after adjusting for multiple comparisons, we found that serum phosphate correlated directly with serum PTH (r = 0.62, P < 0.01) in patients with mild to moderate CRF (creatinine < or = 3.0 mg/dL), independent of serum calcium and 1,25-dihydroxyvitamin D levels. In patients with more severe renal failure (creatinine > 3.0 mg/dL), only the serum calcium correlated with serum PTH (r = -0.47, P < 0.01). While serum 1 ,25-dihydroxyvitamin D showed no correlations with PTH, phosphate, or calcium at any stage of renal failure, the mean 1,25-dihydroxyvitamin D level in patients with mild CRF was lower than that in age-matched controls (24 +/- 3 pg/mL v 37 +/- 2 pg/mL; P < 0.01), suggesting that low 1,25-dihydroxyvitamin D was permissive for enhanced PTH secretion. These data demonstrate an independent association of serum phosphate with PTH in patients with CRF and suggest that phosphate may directly enhance PTH secretion in this setting. This study supports recent animal studies showing a direct parathyroid cell effect of phosphate on PTH secretion.

Plasmin degradation of insulin-like growth factor-binding protein-5 (IGFBP-5): regulation by IGFBP-5-(201-218).

Using the major bone insulin-like growth factor-binding protein (IGFBP) IGFBP-5, we took a mechanistic approach in evaluating the role of the heparin-binding domain of IGFBP-5 in regulating plasmin (Pm) proteolysis of IGFBP-5. Using synthetic IGFBP-5 peptide fragments, we determined that the heparin-binding domain, IGFBP-5-(208-218), inhibits Pm proteolysis of intact IGFBP-5. The mechanism of action of IGFBP-5-(201-218) was by inhibiting Pm binding to substrate IGFBP-5. IGFBP-5-(201-218) action was independent of site of proteolysis, fluid, or solid phase interaction. In addition, IGFBP-5-(201-218) was found to inhibit plasminogen (Pg) activation to Pm IGFBP-5-(201-218) did not directly inhibit the activity of Pm, urokinase Pg activator (PA), or tissue-type PA but acted as a competitive inhibitor of Pg activation by PA, which is in contrast to the stimulating effect of heparin on Pg activation. These data indicate that the heparin-binding domain contains the serine protease (Pg-to-Pm) binding site region of IGFBP-5, and that this region, which is presumed to represent a Pm-induced proteolytic product of IGFBP-5, is capable of regulating Pm action.

Insulin-like growth factor (IGF)-binding protein-5-(201-218) region regulates hydroxyapatite and IGF-I binding.

Insulin-like growth factor-binding protein-5 (IGFBP-5), the major bone IGFBP, modifies the biological activity of IGFs within the osteoblastic pericellular environment. Because glycosaminoglycans modulate IGFBP-5 binding to osteoblast organic extracellular matrix (ECM), we assessed whether the heparin binding domain of IGFBP-5, IGFBP-5-(102-218), modifies the interaction of IGFBP-5 with the inorganic bone ECM hydroxyapatite (HA). Synthetic IGFBP-5-(201-218) peptide increased the binding of IGFBP-5 to HA as well as the binding of IGF-I to HA-bound IGFBP-5. This action was specific for the heparin-binding domain, because IGFBP-5-(130-138), IGFBP-5-(138-152), and IGFBP-5-(1-169) were without effect. IGFBP-5-(201-218) was found to bind directly to IGFBP-5 and cause a threefold enhancement of the IGF-I binding affinity for IGFBP-5, whether IGFBP-5 was bound to HA or was in a matrix-free fluid phase. Heparin inhibited the binding of IGFBP-5 to HA and blocked the interaction of IGFBP-5 with IGFBP-5-(201-218) in the fluid phase, suggesting that the primary heparin-binding domain of IGFBP-5 specifically enhances the binding of IGFBP-5 to HA and increases IGF-I binding to IGFBP-5.

Heparin binding domain of insulin-like growth factor binding protein-5 stimulates mesangial cell migration.

Insulin-like growth factor I (IGF-I) binding protein-5 (IGFBP-5) is produced by mesangial cells (MCs) and likely functions to modulate glomerular IGF-I activity. Although IGFBP-5 may be inhibitory for IGF-stimulated MC activity, preliminary studies suggested that IGFBP-5 acts directly on MCs. To investigate this further, we evaluated the effects of IGFBP-5 on rat MC migration. We found that the carboxytruncated fragment, IGFBP-5-(1-169), inhibited IGF-I-stimulated migration, but intact IGFBP-5 simulated migration when IGF-I was not present. Demonstration that 125I-labeled IGFBP-5 directly binds to MCs further supports an independent role for IGFBP-5. Because heparin inhibited MC binding of 125I-IGFBP-5, we tested the heparin binding peptide, IGFBP-5-(201-218), for stimulatory activity. IGFBP-5-(201-218) stimulated MC migration, and this effect was inhibited by heparin. Because the disintegrin, kistrin, blocked IGF-I-induced migration but not migration induced by IGFBP-5-(201-218), the migratory induction mechanism for the two peptides is different. These data indicate that separate, specific regions of IGFBP-5 are responsible for interactive effects with IGF-I as well as direct effects on MC activity.

Growth hormone receptor activity is stimulated by insulin-like growth factor binding protein 5 in rat osteosarcoma cells.

Osteoblast-like UMR-106.01 rat osteosarcoma cells express high affinity growth hormone (GH) receptors (GHRs). Because osteoblasts secrete insulin-like growth factor binding protein-5 (IGFBP-5), we evaluated whether it also modulates GH binding and GHR expression in UMR cells. Human recombinant intact IGFBP-5 stimulated 125I-hGH binding in a dose-dependent manner (dose range 300-3000 ng/ml), inducing an increase to 193.6 +/- 2.1% of control binding at 3000 ng/ml (P < 0.001). Carboxy-truncated IGFBP-5 also stimulated GH binding but with less potency (125 +/- 2.7% of control at 3000 ng/ml, P < 0.01). GHRs identified by chemical crosslinking of 125I-hGH to cell monolayers increased after treatment with IGFBP-5 and decreased in response to insulin-like growth factor-I (IGF-I). GHR mRNA levels, as quantitated by a solution hybridization RNAse protection assay, increased up to 3 to 7-fold in a time-dependent manner by intact IGFBP-5 but not by carboxy-truncated IGFBP-5. An antiserum to IGFBP-5 reduced basal GH binding to 56.7 +/- 4.3% of control value at a concentration of 0.5% (P < 0.001), showing that IGFBP-5 produced by the cells is a strong regulator of GH binding. IGFBP-5 antiserum also decreased GH binding to 85.9 +/- 0.9% of IGFBP-5 stimulated value (P < 0.001), showing the specificity of IGFBP-5 stimulation. To determine whether the GHR upregulation was physiologically significant, cell proliferation was evaluated after coincubation of IGFBP-5 with low, non-stimulatory concentrations of GH. IGFBP-5 (1000 ng/ml) induced cell proliferation to 116.2 +/- 3.2% of control levels, and coincubation with hGH at 10 ng/ml induced an increase to 133.3 +/- 0.1% of control levels. We conclude that exogenous and endogenous IGFBP-5 upregulate GHR mRNA levels and GH binding and this interaction potentiates GH-stimulated mitogenesis in osteoblastic cells.