Mitofusin 2 plays a critical role in maintaining the functional integrity of the neuromuscular-skeletal axis.

PURPOSE: Mitofusin 2 (Mfn2) is one of two mitofusins involved in regulating mitochondrial size, shape and function, including mitophagy, an important cellular mechanism to limit oxidative stress. Reduced expression of Mfn2 has been associated with impaired osteoblast differentiation and function and a reduction in the number of viable osteocytes in bone. We hypothesized that the genetic absence of Mfn2 in these cells would increase their susceptibility to aging-associated metabolic stress, leading to a progressive impairment in skeletal homeostasis over time. METHODS: Mfn2 was selectively deleted in vivo at three different stages of osteoblast lineage commitment by crossing mice in which the Mfn2 gene was floxed with transgenic mice expressing Cre under the control of the promoter for Osterix (OSX), collagen1a1, or DMP1 (Dentin Matrix Acidic Phosphoprotein 1). RESULTS: Mice in which Mfn2 was deleted using DMP1-cre demonstrated a progressive and dramatic decline in bone mineral density (BMD) beginning at 10 weeks of age (n = 5 for each sex and each genotype from age 10 to 20 weeks). By 15 weeks, there was evidence for a functional decline in muscle performance as assessed using a rotarod apparatus (n = 3; 2 males/ 1 female for each genotype), accompanied by a decline in lean body mass. A marked reduction in trabecular bone mass was evident on bone histomorphometry, and biomechanical testing at 25 weeks (k/o: 2 male/1 female, control 2 male/2 female) revealed severely impaired femur strength. Extensive regional myofiber atrophy and degeneration was observed on skeletal muscle histology. Electron microscopy showed progressive disruption of cellular architecture, with disorganized sarcomeres and a bloated mitochondrial reticulum. There was also evidence of neurodegeneration within the ventral horn and roots of the lumbar spinal cord, which was accompanied by myelin loss and myofiber atrophy. Deletion of Mfn2 using OSX-cre or Col1a1-cre did not result in a musculoskeletal phenotype. Where possible, male and female animals were analyzed separately, but small numbers of animals in each group limited statistical power. For other outcomes, where sex was not considered, small sample sizes might still limit the strength of the observation. CONCLUSION: Despite known functional overlap of Mfn1 and Mfn2 in some tissues, and their co-expression in bone, muscle and spinal cord, deletion of Mfn2 using the 8 kB DMP1 promoter uncovered an important non-redundant role for Mfn2 in maintaining the neuromuscular/bone axis.

Selective deletion of the receptor for CSF1, c-fms, in osteoclasts results in a high bone mass phenotype, smaller osteoclasts in vivo and an impaired response to an anabolic PTH regimen.

The receptor for Colony Stimulating Factor 1 (CSF1), c-fms, is highly expressed on mature osteoclasts suggesting a role for this cytokine in regulating the function of these cells. Consistent with this idea, in vitro studies have documented a variety of effects of CSF1 in mature osteoclasts. To better define the role of CSF1 in these cells, we conditionally deleted c-fms in osteoclasts (c-fms-OC-/-) by crossing c-fmsflox/flox mice with mice expressing Cre under the control of the cathepsin K promoter. The c-fms-OC-/- mice were of normal weight and had normal tooth eruption. However, when quantified by DXA, bone mass was significantly higher in the spine and femur of female knock out mice and in the femurs of male knock out mice. MicroCT analyses of femurs showed that female c-fms-OC-/- mice had significantly increased trabecular bone mass with a similar trend in males and both sexes demonstrated significantly increased trabecular number and reduced trabecular spacing. Histomorphometric analysis of the femoral trabecular bone compartment demonstrated a trend towards increased numbers of osteoclasts, +26% in Noc/BPm and +22% in OcS/BS in the k/o animals but this change was not significant. However, when the cellular volume of osteoclasts was quantified, the c-fms-OC-/- cells were found to be significantly smaller than controls. Mature osteoclasts show a marked spreading response when exposed to CSF1 in a non-gradient fashion. However, osteoclasts freshly isolated from c-fms-OC-/- mice had a near complete abrogation of this response. C-fms-OC-/- mice treated with (1-34)hPTH 80 ng/kg/d in single daily subcutaneous doses for 29 days showed an attenuated anabolic response in trabecular bone compared to wild-type animals. Taken together, these data indicate an important non-redundant role for c-fms in regulating mature osteoclast function in vivo.

An Unanticipated Role for Sphingosine Kinase-2 in Bone and in the Anabolic Effect of Parathyroid Hormone.

Sphingosine-1-phosphate (S1P) is an anabolic clastokine. Sphingosine kinase (SPHK) is the rate-limiting enzyme in S1P production and has 2 isoforms. To evaluate the roles of SPHK1 and SPHK2 in bone, we examined the skeletal phenotype of mice with selective deletion of SPHK1 in osteoclasts (SPHK1-Oc-/-) and mice in which the SPHK2 gene was deleted in all tissues (SPHK2-/-). SPHK1-Oc-/- had normal bone mass. By contrast, SPHK2-/- female mice had a 14% lower spinal bone mineral density (BMD; P < 0.01) and males a 22% lower BMD at the same site (P < 0.001). SPHK2-/- and control mice were subsequently treated either with daily parathyroid hormone [PTH](1-34) or vehicle for 29 days. The response to PTH was significantly attenuated in the SPHK2-/-mice. The mean femoral bone volume to total volume fraction (BV/TV) increased by 24.8% in the PTH-treated female control animals vs 10.6% in the vehicle-treated female controls (P < 0.01). In contrast, in the SPHK2-/- female mice the difference in femoral trabecular BV/TV at the end of treatment was not significant (20.5 vs13.3%, PTH vs vehicle, P = NS). The anabolic response to PTH was significantly attenuated in the spine of male SPHK2-/- mice (29.7% vs 23.1%, PTH vs vehicle, in controls, P < 0.05; 26.9% vs 19.5% PTH vs vehicle in SPHK2-/- mice, P = NS). The spine responded normally in the SPHK2-/- female mice. Interestingly, suppression of sclerostin was blunted in the SPHK2-/- mice when those animals were treated with an anabolic PTH regimen. We conclude that SPHK2 has an important role in mediating both normal bone remodeling and the anabolic response to PTH.

The contribution of cross-talk between the cell-surface proteins CD36 and CD47-TSP-1 in osteoclast formation and function.

Antibody-mediated blockade of cluster of differentiation 47 (CD47)-thrombospondin-1 (TSP-1) interactions blocks osteoclast formation in vitro and attenuates parathyroid hormone (PTH)-induced hypercalcemia in vivo in mice. Hypercalcemia in this model reflects increased bone resorption. TSP-1 has two cell-associated binding partners, CD47 and CD36. The roles of these two molecules in mediating the effects of TSP1 in osteoclasts are unclear. Osteoclast formation was attenuated but not absent when preosteoclasts isolated from CD47-/- mice were cocultured with WT osteoblasts. Suppressing CD36 in osteoclast progenitors also attenuated osteoclast formation. The hypercalcemic response to a PTH infusion was blunted in CD47-/-/CD36-/- (double knockout (DKO)) female mice but not CD47-/- mice or CD36-/- animals, supporting a role for both CD47 and CD36 in mediating this effect. Consistent with this, DKO osteoclasts had impaired resorptive activity when analyzed in vitro Inhibition of nitric oxide (NO) signaling is known to promote osteoclastogenesis, and TSP-1 suppresses NO production and signaling. An anti-TSP-1 antibody increased NO production in osteoclasts, and the inhibitory effect of anti-TSP-1 on osteoclastogenesis was completely rescued by l-nitroarginine methyl ester (l-NAME), a competitive NO synthase inhibitor. Supportive of an important role for CD36 in mediating the pro-osteoclastogenic effects of TSP-1, engaging CD36 with a synthetic agonist, p907, suppressed NO production in anti-TSP-1-treated cultures, allowing osteoclast maturation to occur. These results establish that CD36 and CD47 both participate in mediating the actions of TSP-1 in osteoclasts and establish a physiologically relevant cross-talk in bone tissue between these two molecules.

Breast cancer-associated gene 3 interacts with Rac1 and augments NF-κB signaling in vitro, but has no effect on RANKL-induced bone resorption in vivo.

Breast cancer-associated gene 3 (BCA3) is a recently identified adaptor protein whose functions are still being defined. BCA3 has been reported to be an important regulator of nuclear factor-κB (NF-κB) signaling. It has also been reported to interact with the small GTPase, Rac1. Consistent with that observation, in the present study, BCA3 was found to interact with nuclear Rac1 in 293 cells and influence NF-κB signaling. Additional experiments revealed that depending on cell type, BCA3 augmented, attenuated or had no effect on NF-κB signaling in vitro. Since canonical NF-κB signaling is a critical downstream target from activated receptor activator of nuclear factor κB (RANK) that is required for mature osteoclast formation and function, BCA3 was selectively overexpressed in osteoclasts in vivo using the cathepsin K promoter and the response to exogenous receptor activator of nuclear factor κB ligand (RANKL) administration was examined. Despite its ability to augment NF-κB signaling in other cells, transgenic animals injected with high-dose RANKL had the same hypercalcemic response as their wild­type littermates. Furthermore, the degree of bone loss induced by a 2-week infusion of low-dose RANKL was the same in both groups. Combined with earlier studies, the data from our study data indicate that BCA3 can affect NF-κB signaling and that BCA3 plays a cell-type dependent role in this process. The significance of the BCA3/NF-κB interaction in vivo in bone remains to be determined.

Deletion of Rac in Mature Osteoclasts Causes Osteopetrosis, an Age-Dependent Change in Osteoclast Number, and a Reduced Number of Osteoblasts In Vivo.

Rac1 and Rac2 are thought to have important roles in osteoclasts. Therefore, mice with deletion of both Rac1 and Rac2 in mature osteoclasts (DKO) were generated by crossing Rac1(flox/flox) mice with mice expressing Cre in the cathepsin K locus and then mating these animals with Rac2(-/-) mice. DKO mice had markedly impaired tooth eruption. Bone mineral density (BMD) was increased 21% to 33% in 4- to 6-week-old DKO mice at all sites when measured by dual-energy X-ray absorptiometry (DXA) and serum cross-linked C-telopeptide (CTx) was reduced by 52%. The amount of metaphyseal trabecular bone was markedly increased in DKO mice, but the cortices were very thin. Spinal trabecular bone mass was increased. Histomorphometry revealed significant reductions in both osteoclast and osteoblast number and function in 4- to 6-week-old DKO animals. In 14- to 16-week-old animals, osteoclast number was increased, although bone density was further increased. DKO osteoclasts had severely impaired actin ring formation, an impaired ability to generate acid, and reduced resorptive activity in vitro. In addition, their life span ex vivo was reduced. DKO osteoblasts expressed normal differentiation markers except for the expression of osterix, which was reduced. The DKO osteoblasts mineralized normally in vitro, indicating that the in vivo defect in osteoblast function was not cell autonomous. Confocal imaging demonstrated focal disruption of the osteocytic dendritic network in DKO cortical bone. Despite these changes, DKO animals had a normal response to treatment with once-daily parathyroid hormone (PTH). We conclude that Rac1 and Rac2 have critical roles in skeletal metabolism.

AgRP Neurons Regulate Bone Mass.

The hypothalamus has been implicated in skeletal metabolism. Whether hunger-promoting neurons of the arcuate nucleus impact the bone is not known. We generated multiple lines of mice to affect AgRP neuronal circuit integrity. We found that mice with Ucp2 gene deletion, in which AgRP neuronal function was impaired, were osteopenic. This phenotype was rescued by cell-selective reactivation of Ucp2 in AgRP neurons. When the AgRP circuitry was impaired by early postnatal deletion of AgRP neurons or by cell autonomous deletion of Sirt1 (AgRP-Sirt1(-/-)), mice also developed reduced bone mass. No impact of leptin receptor deletion in AgRP neurons was found on bone homeostasis. Suppression of sympathetic tone in AgRP-Sirt1(-/-) mice reversed osteopenia in transgenic animals. Taken together, these observations establish a significant regulatory role for AgRP neurons in skeletal bone metabolism independent of leptin action.

The transcription factor T-box 3 regulates colony-stimulating factor 1-dependent Jun dimerization protein 2 expression and plays an important role in osteoclastogenesis.

Colony-stimulating factor 1 (CSF1) is known to promote osteoclast progenitor survival, but its roles in osteoclast differentiation and mature osteoclast function are less well understood. In a microarray screen, Jun dimerization protein 2 (JDP2) was identified as significantly induced by CSF1. Recent reports indicate that JDP2 is required for normal osteoclastogenesis and skeletal metabolism. Because there are no reports on the transcriptional regulation of this gene, the DNA sequence from -2612 to +682 bp (relative to the transcription start site) of the JDP2 gene was cloned, and promoter activity was analyzed. The T box-binding element (TBE) between -191 and -141 bp was identified as the cis-element responsible for CSF1-dependent JDP2 expression. Using degenerate PCR, Tbx3 was identified as the major isoform binding the TBE. Overexpression of Tbx3 induced JDP2 promoter activity, whereas suppressing Tbx3 expression substantially attenuated CSF1-induced transcription. Suppressing Tbx3 in osteoclast precursors reduced JDP2 expression and significantly impaired RANKL/CSF1-induced osteoclastogenesis. A MEK1/2-specific inhibitor was found to block CSF1-induced JDP2 expression. Consistent with these data, JDP2(-/-) mice were found to have increased bone mass. In summary, CSF1 up-regulates JDP2 expression by inducing Tbx3 binding to the JDP2 promoter. The downstream signaling cascade from activated c-Fms involves the MEK1/2-ERK1/2 pathway. Tbx3 plays an important role in osteoclastogenesis at least in part by regulating CSF1-dependent expression of JDP2.

Measurement of plasma, serum, and platelet serotonin in individuals with high bone mass and mutations in LRP5.

It has recently been suggested that the low-density lipoprotein receptor-related protein 5 (LRP5) regulates bone mass by suppressing secretion of serotonin from duodenal enterochromaffin cells. In mice with targeted expression of a high bone mass-causing (HBM-causing) LRP5 mutation and in humans with HBM LRP5 mutations, circulating serotonin levels have been reported to be lower than in controls whereas individuals with loss-of-function mutations in LRP5 have high blood serotonin. In contrast, others have reported that conditionally activating a knock-in allele of an HBM-causing LRP5 mutation in several tissues, or genetic deletion of LRP5 in mice has no effect on serum serotonin levels. To further explore the possible association between HBM-causing LRP5 mutations and circulating serotonin, levels of the hormone were measured in the platelet poor plasma (PPP), serum, and platelet pellet (PP) of 16 affected individuals from 2 kindreds with HBM-causing LRP5 mutations (G171V and N198S) and 16 age-matched controls. When analyzed by HPLC, there were no differences in levels of serotonin in PPP and PP between affected individuals and age-matched controls. Similarly, when analyzed by ELISA, there were no differences in PPP or PP between these two groups. By ELISA, serum levels of serotonin were higher in the affected individuals when compared to age-matched controls. A subgroup analysis of only the G171V subjects (n=14) demonstrated that there were no differences in PPP and PP serotonin between affected individuals and controls when analyzed by HPLC. PP serotonin was lower in the affected individuals when measured by ELISA but serum serotonin levels were not different. We conclude that there is no change in PPP serotonin in individuals with HBM-causing mutations in LRP5.

Increasing dietary protein acutely augments intestinal iron transporter expression and significantly increases iron absorption in rats.

Iron (Fe) deficiency is endemic worldwide. Little data are available regarding acute effects of dietary protein on intestinal Fe absorption. The current study evaluated the short-term effects of increasing dietary protein on Fe absorption and expression of genes involved in Fe homeostasis. Sprague Dawley rats (24, female) were randomly assigned to custom-formulated isocaloric diets containing 40, 20 (control), or 5% protein (as percentage of total kilocalories) for 7 d. Whole-body Fe balance studies demonstrated that Fe retention was greater in the 40% group than in the 5% group (30.8 vs. 7.3%; P<0.01). In a separate study utilizing stable iron isotopes, the 40% group absorbed 30% of ingested Fe, while the 20% group absorbed 18% (P=0.005). Whole-genome profiling revealed that increasing dietary protein from 5 to 40% increased duodenal transcript expression of divalent metal transporter 1 (DMT1) 3.2-fold, duodenal cytochrome b (Dcytb) 1.8-fold, and transferrin receptor (TfR) 1.8-fold. Consistent with these findings, DMT1 transcript expression was 4-fold higher in RNA prepared from duodenal mucosa in the 40% group compared to the 20% group (P<0.001). These data suggest that increasing dietary protein increases intestinal Fe absorption in part by up-regulating DMT1, Dcytb, and TfR.

The effect of dietary protein on intestinal calcium absorption in rats.

Increasing dietary protein intake in humans acutely increases urinary calcium. Isotopic absorption studies have indicated that, at least in the short term, this is primarily due to increased intestinal Ca absorption. To explore the mechanisms underlying dietary protein's effect on intestinal Ca absorption, female Sprague Dawley rats were fed a control (20%), low (5%), or high (40%) protein diet for 7 d, and Ca balance was measured during d 4-7. On d 7, duodenal mucosa was harvested and brush border membrane vesicles (BBMVs) were prepared to evaluate Ca uptake. By d 7, urinary calcium was more than 2-fold higher in the 40% protein group compared with control (4.2 mg/d vs. 1.7 mg/d; P < 0.05). Rats consuming the 40% protein diet both absorbed and retained more Ca compared with the 5% protein group (absorption: 48.5% vs. 34.1% and retention: 45.8% vs. 33.7%, respectively; P < 0.01). Ca uptake was increased in BBMVs prepared from rats consuming the high-protein diet. Maximum velocity (V(max)) was higher in the BBMVs prepared from the high-protein group compared with those from the low-protein group (90 vs. 36 nmol Ca/mg protein x min, P < 0.001; 95% CI: 46-2486 and 14-55, respectively). The Michaelis Menten constant (K(m)) was unchanged (2.2 mm vs. 1.8 mm, respectively; P = 0.19). We conclude that in rats, as in humans, acute increases in protein intake result in hypercalciuria due to augmented intestinal Ca absorption. BBMV Ca uptake studies suggest that higher protein intake improves Ca absorption, at least in part, by increasing transcellular Ca uptake.

Dominant role of CD47-thrombospondin-1 interactions in myeloma-induced fusion of human dendritic cells: implications for bone disease.

Lytic bone disease in myeloma is characterized by an increase in multinucleate osteoclasts in close proximity to tumor cells. However, the nature of osteoclast precursors and the mechanisms underlying multinuclearity are less understood. Here we show that culture of myeloma cell lines as well as primary myeloma cells with human dendritic cells (DCs) but not monocytes or macrophages leads to spontaneous cell-cell fusion, which then leads to the facile formation of multinucleate bone-resorbing giant cells. Osteoclastogenesis is cell contact dependent, leading to up-regulation of thrombospondin-1 (TSP-1) in DCs. Disruption of CD47-TSP-1 interaction by TSP-1-blocking antibodies or down-regulation of CD47 on tumor cells by RNA interference abrogates tumor-induced osteoclast formation. Blockade of CD47-TSP-1 interactions also inhibits receptor activator for nuclear factor kappaB ligand- and macrophage colony-stimulating factor-induced formation of osteoclasts from human monocytes. Further, TSP-1 blockade attenuates hypercalcemia induced by parathyroid hormone in vivo. These data point to a role for CD47-TSP-1 interactions in regulating cell-fusion events involved in human osteoclast formation. They also suggest that DCs, known to be enriched in myeloma tumors, may be direct precursors for tumor-associated osteoclasts. Disruption of CD47-TSP-1 interactions or preventing the recruitment of DCs to tumors may provide novel approaches to therapy of myeloma bone disease and osteoporosis.

Targeted overexpression of the two colony-stimulating factor-1 isoforms in osteoblasts differentially affects bone loss in ovariectomized mice.

Colony-stimulating factor-1 (CSF1) is one of two cytokines required for normal osteoclastogenesis. There are two major isoforms of CSF1, the cell-surface or membrane-bound isoform (mCSF1) and soluble CSF1 (sCSF1). Whether these isoforms serve nonredundant functions in bone is unclear. To explore this question, we generated transgenic mice expressing human sCSF1, human mCSF1, or both (s/mCSF1) in osteoblasts using the 2.3-kb rat alphaI-collagen promoter. Bone density determined by peripheral quantitative computed tomography was significantly reduced in mCSF1, sCSF1, and s/mCSF1 transgenic mice compared with wild-type animals. When analyzed by sex, sCSF1, and s/mCSF1, female animals but not mCSF1 female mice were found to have greater bone loss than their male littermates (-20 vs. -9.2%; P<0.05 for sCSF1 and -21.6 vs. -11.2% for s/mCSF1; P<0.01). By breeding CSF1 isoform-selective transgenic mice to an op/op background, mice were generated in which a single CSF1 isoform was the only source of the cytokine (sCSF1op/op and mCSF1op/op). Unlike osteoblast-targeted overexpression of mCSF1, selective transgenic expression of sCSF1 did not completely correct the op/op phenotype in 5-mo-old animals. Interestingly, compared with sham-ovariectomized mice of the same genotype, ovariectomy in sCSF1op/op mice led to a greater loss of spinal bone mineral density (22.1%) than was seen in either mCSF1op/op mice (12.9%) or in wild-type animals (10.9%). Our findings support the conclusion that sCSF1 and mCSF1 serve nonredundant functions in bone and that sCSF1 may play a role in mediating estrogen-deficiency bone loss.

The anabolic response to parathyroid hormone is augmented in Rac2 knockout mice.

PTH is the only currently available anabolic therapy for osteoporosis. In clinical practice, the skeletal response to PTH varies and because therapy is limited to 2 yr, approaches to maximize the therapeutic response are desirable. Rac2 is a small GTPase that is expressed only in hematopoietic tissue. Rac2(-/-) mice have a slight increase in bone mass and osteoclasts isolated from these animals have reduced basal resorptive activity and reduced chemotaxis. To evaluate the anabolic response to PTH in Rac2(-/-) mice, we treated 18 Rac2(-/-) and 17 control, age-matched wild-type animals once daily for 28 d with 80 ng/g body weight of h(1-34)PTH. Treatment resulted in significantly greater increments in spinal, femur, and total bone density in the Rac2(-/-) as compared with wild-type animals. Microcomputed tomography analysis demonstrated greater increases in trabecular thickness and cortical thickness in the knockout mice. Interestingly, histomorphometric analysis showed an equivalent increase in osteoblast and osteoclast number in response to PTH treatment in both groups of animals. However, as judged by changes in serum markers, the resorptive response to PTH was impaired. Thus, telopeptide of type 1 collagen was 15.9+/-6.9 ng/ml after PTH treatment in the knockout animals and 26.8+/-11.1 ng/ml in the PTH-treated wild-type group. In contrast, serum aminoterminal propeptide of type 1 collagen and osteocalcin were equivalent in both groups. We conclude that, in the genetic absence of Rac2, the anabolic response to PTH is increased. This appears to be due to attenuated resorptive activity of osteoclasts.

Expression and synthesis of bone morphogenetic proteins by osteoclasts: a possible path to anabolic bone remodeling.

Skeletal remodeling is a finely orchestrated process coupling bone formation to bone resorption. The dynamics of coupling is regulated by the microenvironment at the bone remodeling site, which in turn is influenced by the intercellular communication between cells like osteoclasts and osteoblasts. Understanding the dynamics of coupling is important in devising new therapeutic approaches to the treatment of skeletal diseases characterized by disturbances in the bone remodeling process. In this study, we report the localization of bone morphogenetic proteins (BMPs) in osteoclasts generated from primary cocultures of bone marrow cells from mouse femur and tibia with mouse calvarial osteoblasts, using immunocytochemistry and in situ hybridization. Positive staining was seen in osteoclasts for BMP-2, -4, -6, and -7. Real-time PCR was used to quantitatively confirm the expression of transcripts for BMP-2, BMP-4, and BMP-6 mRNA in murine osteoclasts. Finally, the presence of BMP-2, -4, -6, and-7 proteins was confirmed in osteoclast lysates by Western blotting. Overall, our data suggest a possible direct role for osteoclasts in promoting bone formation via expression and synthesis of BMPs, which then would play an important role in promoting the recruitment, proliferation, and differentiation of osteoblasts at bone resorption sites.

[Weed biodiversity in winter wheat field of loess soil under different fertilization regimes].

Employing an inverted 'W' investigation procedure with 9 sampling locations and adopting a biodiversity analysis approach integrated with typical statistic method, this paper studied the effects of different long-term stationary fertilization regimes on the weed biodiversity in winter wheat fields on loess soil. The results showed that in the experimental plots, there were 16 weed species belonging to 10 family and 16 genera, occupying about 34% of the total number of weed species in winter wheat fields in Shaanxi Province. The weed biodiversity was decreased with the improvement of soil nutrient status. There were 3-5 weed populations in treatments NPK and NPK plus organic materials, and 6-8 populations in treatments CK, N, NK and NP. The relative abundance of weeds ranged from 0 to 73%, and the ranges of Shannon's diversity index, Shannon's evenness index and Margalef' s species richness index were 0.2-1.08, 0.05-0.26 and 0.26-1.26, respectively. All of these 3 parameters were higher in unbalanced than in balanced fertilization treatments, and the differences between unbalanced and balanced fertilization treatments were significant in most case, which was probably due to the different status of available soil nutrients and might have different effects on the growth of weeds.

The effect of aging on the skeletal response to intermittent treatment with parathyroid hormone.

Little is known about the modifying effects of age on the skeletal response to intermittent treatment with PTH. We therefore compared the response of 63 aged (18 month old) and 61 young-adult (3 month old) C57BL/6 mice to 4 wk of daily sc injections of either vehicle or h(1-34)PTH at a dose of 95 ng/g body weight. The increase in total body bone mineral density (BMD), compared with vehicle-treated animals, was similar in aged and young-adult mice (+5.6 vs. +6.3%). Aged animals demonstrated a greater increase in spinal BMD than their younger counterparts (+12.0 vs. +5.1%, P = 0.01; absolute increment: 57 x 10(-4) vs. 28 x 10(-4) g/cm(2)). Microcomputed tomography analyses in a subset of the vertebrae showed a trend toward higher L5 trabecular bone volume fraction in the PTH-treated aged animals (+40.2 vs. +19.6%). Vertebral histomorphometry demonstrated a greater PTH-induced increase in osteoblast number in aged vs. young-adult animals (694 vs. 396 cells/mm(2)). In contrast, in the femur the PTH-induced increase in BMD tended to be greater in the young-adult than the aged animals, although this did not reach statistical significance (8.1 vs. 4.2%). The numbers of osteoblast progenitors and mineralizing colonies in cultured marrow were unaffected by PTH treatment in either group. We conclude that aging differentially impacts the regional skeletal response to PTH such that the increase in BMD in the spine is augmented, whereas that in the femur is unaffected. Effects on osteoblast progenitor recruitment do not seem to be the basis for these changes.

The cell surface form of colony-stimulating factor-1 is biologically active in bone in vivo.

The specific biological function of the cell surface or membrane-bound isoform of colony-stimulating factor-1 (mCSF-1) is not well understood. To help define the role of this isoform in bone, we developed a transgenic mouse in which targeted expression of human mCSF-1 in osteoblasts was achieved under the control of the 2.4-kb rat collagen type I alpha promoter. Bone density, determined by peripheral quantitative computed tomography, was reduced 7% in mCSF-1 transgenic compared with that in wild-type mice. Histomorphometric analyses indicated that the number of osteoclasts in bone (NOc/BPm, NOc/TAR, OcS/BS) was significantly increased in transgenic mice (1.7- to 1.8-fold; P < 0.05 to P < 0.01) compared with that in wild-type animals. Interestingly, the osteoblast-restricted isoform transgene corrected the osteopetrosis seen in CSF-1-deficient op/op mice. Skeletal growth and bone density in op/op mice expressing mCSF-1 in osteoblasts were similar to those in wild-type mice and were dramatically different from those in the unmanipulated op/op animals. The op/op mice expressing mCSF-1 in bone had normal incisor and molar tooth eruption, whereas the op/op mice evidenced the expected failure of tooth eruption. These findings directly support the conclusion that mCSF-1 is functionally active in bone in vivo and is probably an important local source of CSF-1.