Citations

Abstract To examine the functional role of membrane-bound SLF, we evaluated the growing skeletons of WT and SLF mutant (Sl/Sld) mice that do not produce this protein using DXA, bone histomorphometry, cell culture, and flow cytometry. Deletion of membrane-bound SLF delays bone growth, decreases bone mass and BMD, impairs osteoblast function, and increases osteoclast surface in young mice. Introduction: Mutations at the murine steel locus lead to a defect in the development of hematopoietic stem cells, mast cells, and germ cells. Two isoforms of steel factor (SLF), soluble and membrane-associated, have been reported. Soluble SLF increases osteoclast formation and activity in cell culture. The effects of deletion of membrane-bound SLF on bone metabolism in mice have yet to be determined and are the subject of this study. Materials and Methods: Five-, 7-, and 12-week-old male and 5-week-old female WCB6F1/J-KitlSl/KitlSl-d (Sl/Sld) mice and wildtype (WT) littermates were used. BMD and bone mass, growth, architecture, and turnover were evaluated by DXA (males and females) and histomorphometry (males only). Primary osteoblasts isolated from humeri of 5-week-old male WT and Sl/Sld mice were used to determine osteoblast function, and bone marrow cells from tibias and femurs of these mice were analyzed to determine cell surface expression of osteoclast precursors. Results and Conclusions: Young Sl/Sld mice grew more slowly, had a reduced bone mass, and had shorter bones than WT littermates. Male mutants had significantly decreased whole body BMD in all age groups, largely because of a reduction in BMC. Tibial cross-sectional, cortical, and marrow area of cortical bone and cancellous bone volume was reduced in the mutants at all ages. The osteopenia in Sl/Sld was caused by increased osteoclast surface at all ages and decreased osteoblast surface at 5 weeks of age. [3H]thymidine incorporation studies showed that proliferation of osteoblasts derived from mutant mice was significantly suppressed (56%). Moreover, a decrease in mineralization was observed in Sl/Sld osteoblast culture. Fluorescence-activated cell sorting analysis of bone marrow cells from Sl/Sld mice revealed a 65% increase in the percentage of c-Fms+CD11b+RANK+ cells compared with WT controls. These findings suggest that membrane-bound SLF/c-Kit signaling plays a role in the regulation of peak bone mass.

Abstract We examined the time course effects of continuous PTH on cortical bone and mechanical properties. PTH increased cortical bone turnover and induced intracortical porosity with no deleterious effect on bone strength. Withdrawal of PTH increased maximum torque to failure and stiffness with no change in energy absorbed. Introduction: The skeletal response of cortical bone to parathyroid hormone (PTH) is complex and species dependent. Intermittent administration of PTH to rats increases periosteal and endocortical bone formation but has no known effects on intracortical bone turnover. The effects of continuous PTH on cortical bone are not clearly established. Materials and Methods: Eighty-four 6-month-old female Sprague-Dawley rats were divided into three control, six PTH, and two PTH withdrawal (WD) groups. They were subcutaneously implanted with osmotic pumps loaded with vehicle or 40 μg/kg BW/day human PTH(1-34) for 1, 3, 5, 7, 14, and 28 days. After 7 days, PTH was withdrawn from two groups of animals for 7 (7d-PTH/7d-WD) and 21 days (7d-PTH/21d-WD). Histomorphometry was performed on periosteal and endocortical surfaces of the tibial diaphysis in all groups. μCT of tibias and mechanical testing by torsion of femora were performed on 28d-PTH and 7d-PTH/21d-WD animals. Results and Conclusions: Continuous PTH increased periosteal and endocortical bone formation, endocortical osteoclast perimeter, and cortical porosity in a time-dependent manner, but did not change the mechanical properties of the femur, possibly because of addition of new bone onto periosteal and endocortical surfaces. Additionally, withdrawal of PTH restored normal cortical porosity and increased maximum torque to failure and stiffness. We conclude that continuous administration of PTH increased cortical porosity in rats without having a detrimental effect on bone mechanical properties.

Abstract Exuberant inflammatory responses are associated with respiratory failure during Pneumocystis pneumonia. Alveolar epithelial cells (AECs) promote Pneumocystis attachment and proliferation, but also contribute prominently to host cytokine-mediated inflammation during pneumonia. Recent investigations indicate that AECs produce macrophage inflammatory protein-2 (MIP-2) and tumor necrosis factor-α (TNF-α) following challenge with Pneumocystis carinii. Nuclear factor-κB (NF-κB) is a ubiquitous transcription factor critical for regulation of proinflammatory cytokine expression. Herein, we assess rat AEC NF-κB responses to challenge with a P. carinii β-glucan cell wall component (PCBG). Prominent nuclear translocation of p65 NF-κB was demonstrated following PCBG challenge. NF-κB activation was in part mediated through Protein Kinase C (PKC) signaling pathways. PCBG challenge of AECs was also shown to induce MIP-2 and TNF-α mRNA production, a response that was ameliorated by NF-κB inhibition. MIP-2 protein expression was also dramatically increased by PCBG challenge, in a manner that was significantly attenuated by both PKC and NF-κB inhibition. The data further demonstrate that AEC chemokine responses were not mediated by the recently described dectin-1 receptor, but instead involved participation of cell surface lactosylceramide. These data support a significant role for AECs in host responses during Pneumocystis pneumonia, and further indicate that β-glucan induces inflammatory cytokine production through NF-κB–dependent mechanisms.

Abstract To test the clinical hypothesis that an estrogen-enriched milieu enhances GHRH action, we administered placebo (Pl) and estradiol-17β (E2) orally for 23 d to six postmenopausal women in a prospectively randomized, double-masked, within-subject crossover design with 6 wk intervening. The GHRH stimulation protocol entailed consecutive iv infusion of l-arginine and a single iv pulse of saline or one of five randomly ordered doses of recombinant human GHRH-1,44-amide (0.03, 0.1, 0.3, 1.0, or 3.0 μg/kg) in a total of 12 separate morning, fasting sessions. GH secretion was monitored by sampling blood every 10 min for 6 h; chemiluminescence assay of GH concentrations; deconvolution analysis of stimulated GH release; and nonlinear dose-response reconstruction. Supplementation with E2, compared with Pl: 1) increased (mean ± sem) E2 concentrations from 18 ± 3 (Pl) to 164 ± 12 pg/ml (to convert to picomoles per liter, multiply by 3.57) (P < 0.001); 2) decreased IGF-I concentrations from 181 ± 14 to 120 ± 11 μg/liter (P < 0.01); 3) elevated mean GH concentrations from 0.27 ± 0.06 to 0.59 ± 0.08 μg/liter (P = 0.014); 4) potentiated GH secretion stimulated by l-arginine alone by 1.43-fold (P = 0.012); 5) reduced the ED50 of GHRH from 0.27 ± 0.02 to 0.13 ± 0.01 μg/kg (P < 0.01), denoting enhanced GHRH potency; and 6) heightened the maximal slope of the dose-response function from 1.1 ± 0.1 to 1.4 ± 0.05 [(μg/liter) (μg/kg)−1] (P < 0.05), signifying augmented pituitary sensitivity. The foregoing facilitative mechanisms were specific because E2 replacement did alter maximal l-arginine/GHRH-induced GH secretion, indicating unchanged secretagogue efficacy. In conclusion, inasmuch as E2 also attenuates inhibition of GH secretion by infused somatostatin and potentiates stimulation of GH secretion by GH-releasing peptide-2, we postulate that estrogenic steroids drive pulsatile GH production in part via mechanisms that include all three of GHRH, somatostatin, and putatively GH-releasing peptide/ghrelin signaling.

The effect of aging on long bone mechanical properties and bone formative capacity was characterized in the male Fisher 344 rat. The femurs of rats from three age groups (4 mo., 12 mo. and 28 mo.) were tested in three-point bending to determine their structural properties. The apparent material properties were then calculated by adjusting for bone geometry. Bone formation was assessed by dynamic histomorphometry of both cortical and cancellous bone as well as by Northern blot analysis for the expression of the osteoblast phenotypic proteins osteopontin (OP), osteocalcin (OC), type I collagen (COL) and alkaline phosphatase (AP). Aging resulted in a decline in the apparent material properties that was associated with a compensatory alteration of bone geometry that preserved structural strength and stiffness. Histomorphometric analysis revealed significant age-related decreases in cancellous bone volume, trabecular number and increased trabecular separation suggesting the existence of senile osteopenia in the proximal tibia of the male Fisher 344 rat. A significant decline in bone formation rate (BFR), but not mineral apposition rate, suggests that a reduction in osteoblast number, but not osteoblast activity, contributes to age-related bone loss. The decline in BFR with aging was reflected in a decreased mRNA expression for OP, OC and COL but not AP. Further, the pattern of mRNA expression was consistent with reduced osteoblast differentiation with aging. The present study indicates the age-related decline in material properties of long bones is paralleled by a decrease in osteogenesis.