GmMPK6 Positively Regulates Salt Tolerance through Induction of GmRbohI1 in Soybean.

Salt stress is a critical environmental stress that impairs plant growth and development, especially in crop productivity; therefore, understanding the salt response in plants is the basis for their development of salt tolerance. Under salinity, soybean mitogen-activated protein kinase 6 (GmMPK6) is activated and positively regulates reactive oxygen species (ROS) generation. However, it is not yet elucidated how GmMPK6 regulates ROS generation and its role in salt tolerance. Here, we show that GmMPK6, solely activated in NaCl treatment, and gene expression of GmRbohI1 was not only reduced by MPK inhibitor SB202190 in NaCl treatment, but also increased in a GMKK1-expressing protoplast. Furthermore, SB202190 and the NADPH-oxidase inhibitor, diphenyleneiodonium chloride, increased susceptibility to salt stress. The expression of GmRD19A was induced by NaCl treatment, but this expression was compromised by SB202190. Consequently, we revealed that GmMPK6 induces ROS generation through the transcriptional regulation of GmRbohI1 and increases salt tolerance in soybean.

Organization of nif gene cluster in Frankia sp. EuIK1 strain, a symbiont of Elaeagnus umbellata.

The nucleotide sequence of a 20.5-kb genomic region harboring nif genes was determined and analyzed. The fragment was obtained from Frankia sp. EuIK1 strain, an indigenous symbiont of Elaeagnus umbellata. A total of 20 ORFs including 12 nif genes were identified and subjected to comparative analysis with the genome sequences of 3 Frankia strains representing diverse host plant specificities. The nucleotide and deduced amino acid sequences showed highest levels of identity with orthologous genes from an Elaeagnus-infecting strain. The gene organization patterns around the nif gene clusters were well conserved among all 4 Frankia strains. However, characteristic features appeared in the location of the nifV gene for each Frankia strain, depending on the type of host plant. Sequence analysis was performed to determine the transcription units and suggested that there could be an independent operon starting from the nifW gene in the EuIK strain. Considering the organization patterns and their total extensions on the genome, we propose that the nif gene clusters remained stable despite genetic variations occurring in the Frankia genomes.

Postembryonic seedling lethality in the sterol-deficient Arabidopsis cyp51A2 mutant is partially mediated by the composite action of ethylene and reactive oxygen species.

Seedling-lethal phenotypes of Arabidopsis (Arabidopsis thaliana) mutants that are defective in early steps in the sterol biosynthetic pathway are not rescued by the exogenous application of brassinosteroids. The detailed molecular and physiological mechanisms of seedling lethality have yet to be understood. Thus, to elucidate the underlying mechanism of lethality, we analyzed transcriptome and proteome profiles of the cyp51A2 mutant that is defective in sterol 14alpha-demethylation. Results revealed that the expression levels of genes involved in ethylene biosynthesis/signaling and detoxification of reactive oxygen species (ROS) increased in the mutant compared with the wild type and, thereby, that the endogenous ethylene level also increased in the mutant. Consistently, the seedling-lethal phenotype of the cyp51A2 mutant was partly attenuated by the inhibition of ethylene biosynthesis or signaling. However, photosynthesis-related genes including Rubisco large subunit, chlorophyll a/b-binding protein, and components of photosystems were transcriptionally and/or translationally down-regulated in the mutant, accompanied by the transformation of chloroplasts into gerontoplasts and a reduction in both chlorophyll contents and photosynthetic activity. These characteristics observed in the cyp51A2 mutant resemble those of leaf senescence. Nitroblue tetrazolium staining data revealed that the mutant was under oxidative stress due to the accumulation of ROS, a key factor controlling both programmed cell death and ethylene production. Our results suggest that changes in membrane sterol contents and composition in the cyp51A2 mutant trigger the generation of ROS and ethylene and eventually induce premature seedling senescence.

Nuclear Translocation of Soybean MPK6, GmMPK6, Is Mediated by Hydrogen Peroxide in Salt Stress.

The plant mitogen-activated protein kinase (MPK) cascade, a highly conserved signal transduction system in eukaryotes, plays a crucial role in the plant's response to environmental stimuli and phytohormones. It is well-known that nuclear translocation of MPKs is necessary for their activities in mammalian cells. However, the mechanism underlying nuclear translocation of plant MPKs is not well elucidated. In the previous study, it has been shown that soybean MPK6 (GmMPK6) is activated by phosphatidic acid (PA) and hydrogen peroxide (H2O2), which are two signaling molecules generated during salt stress. Using the two signaling molecules, we investigated how salt stress triggers its translocation to the nucleus. Our results show that the translocation of GmMPK6 to the nucleus is mediated by H2O2, but not by PA. Furthermore, the translocation was interrupted by diphenylene iodonium (DPI) (an inhibitor of RBOH), confirming that H2O2 is the signaling molecule for the nuclear translocation of GmMPK6 during salt stress.

Differential expression of three catalase genes in the small radish (Rhaphanus sativus L. var. sativus).

Three catalase cDNA clones were isolated from the small radish (Raphanus sativus L.). Their nucleotide and deduced amino acid sequences showed the greatest homology to those of Arabidopsis. Genomic Southern blot analysis, using RsCat1 cDNA as a probe, showed that catalases are encoded by small multigene family in the small radish. Nondenaturing polyacrylamide gels revealed the presence of several catalase isozymes, the levels of which varied among the organs examined. The isozyme activities were assigned the individual catalase genes by Northern analysis using total RNA from different organs. The three catalase genes were differentially expressed in response to treatments such as white light, xenobiotics, osmoticum, and UV. Their expression in seedlings was controlled by the circadian clock under a light/dark cycle and/or in constant light. Interestingly, RsCat1 transcripts peaked in the morning, while those of RsCat2 and RsCat3 peaked in the early evening. Our results suggest that the RsCat enzymes are involved in defense against the oxidative stress induced by environmental changes.

Expression of a functional type-I chalcone isomerase gene is localized to the infected cells of root nodules of Elaeagnus umbellata.

A putative type-I chalcone isomerase (CHI) cDNA clone EuNOD-CHI was previously isolated from the root nodule of Elaeagnus umbellata [Kim et al. (2003)]. To see if it encodes a functional CHI, we ectopically overexpressed it in the Arabidopsis (Arabidopsis thaliana) transparent testa 5 (tt5) mutant, which is defective in naringenin production and has yellow seeds due to proanthocyanidin deficiency. Ectopic overexpression of EuNOD-CHI resulted in recovery of normal seed coat color. Naringenin produced by CHI from naringenin chalcone was detected in the transgenic lines like in the wild-type, whereas it was absent from the tt5 mutant. We conclude that EuNOD-CHI encodes a functional type-I CHI. In situ hybridization revealed that EuNOD-CHI expression is localized to the infected cells of the fixation zone in root nodules.

Expression of EuNOD-ARP1 encoding auxin-repressed protein homolog is upregulated by auxin and localized to the fixation zone in root nodules of Elaeagnus umbellata.

Root nodule formation is controlled by plant hormones such as auxin. Auxin-repressed protein (ARP) genes have been identified in various plant species but their functions are not clear. We have isolated a full-length cDNA clone (EuNOD-ARP1) showing high sequence homology to previously identified ARP genes from root nodules of Elaeagnus umbellata. Genomic Southern hybridization showed that there are at least four ARP-related genes in the genome of E. umbellata. The cDNA clone encodes a polypeptide of 120 amino acid residues with no signal peptide or organelle-targeting signals, indicating that it is a cytosolic protein. Its cytosolic location was confirmed using Arabidopsis protoplasts expressing a EuNOD-ARP1:smGFP fusion protein. Northern hybridization showed that EuNOD-ARP1 expression was higher in root nodules than in leaves or uninoculated roots. Unlike the ARP genes of strawberry and black locust, which are negatively regulated by exogenous auxin, EuNOD-ARP1 expression is induced by auxin in leaf tissue of E. umbellata. In situ hybridization revealed that EuNOD-ARP1 is mainly expressed in the fixation zone of root nodules.

Overexpression in Arabidopsis of a plasma membrane-targeting glutamate receptor from small radish increases glutamate-mediated Ca2+ influx and delays fungal infection.

Ionotropic glutamate receptors (iGluRs) are ligand-gated nonselective cation channels that mediate fast excitatory neurotransmission. Although homologues of the iGluRs have been identified in higher plants, their roles are largely unknown. In this work we isolated a full-length cDNA clone (RsGluR) encoding a putative glutamate receptor from small radish. An RsGluR: mGFP fusion protein was localized to the plasma membrane. In Arabidopsis thaliana overexpressing the full-length cDNA, glutamate treatment triggered greater Ca2+ influx in the root cells of transgenic seedlings than in those of the wild type. Transgenic plants exhibited multiple morphological changes such as necrosis at their tips and the margins of developing leaves, dwarf stature with multiple secondary inflorescences, and retarded growth, as previously observed in transgenic Arabidopsis overexpressing AtGluR3.2 [Kim et al. (2001)]. Microarray analysis showed that jasmonic acid (JA)-responsive genes including defensins and JA-biosynthetic genes were up-regulated. RsGluR overexpression also inhibited growth of a necrotic fungal pathogen Botrytis cinerea possibly due to up-regulation of the defensins. Based on these results, we suggest that RsGluR is a glutamate-gated Ca2+ channel located in the plasma membrane of higher plants and plays a direct or indirect role in defense against pathogen infection by triggering JA biosynthesis.

Differential expression of three catalase genes in hot pepper (Capsicum annuum L.).

Three different catalase cDNA clones (CaCat1, CaCat2, and CaCat3) were isolated from hot pepper (Capsicum annuum L.), and their expression patterns were analyzed at the levels of mRNA and enzyme activity. Northern hybridization showed that the three catalase genes were differentially expressed in various organs, and that expression of CaCat1 and CaCat2 was regulated differently by the circadian rhythm. In situ hybridization revealed different spatial distributions of CaCat1 and CaCat2 transcripts in leaf and stem. In response to wounding and paraquat treatment, CaCat1 mRNA increased at 4-12 h in both paraquat-treated and systemic leaves. In contrast, wounding had no significant effect on expression of the catalase genes. The increase of catalase activity in the paraquat-treated and systemic leaves paralleled that of CaCat1 mRNA, but did not match that of CaCat1 mRNA in paraquat-treated stems. Our results suggest that CaCat1 may play a role in responses to environmental stresses.

Differential expression patterns of an acidic chitinase and a basic chitinase in the root nodule of Elaeagnus umbellata.

Two cDNA clones encoding chitinase were isolated from a root nodule cDNA library of Elaeagnus umbellata by the hybridization-competition method. The two clones, EuNOD-CHT1 and EuNOD-CHT2, encode for 335 and 317 amino acid residues with the molecular mass of mature proteins being 33.3 and 31.1 kDa, respectively. The two chitinases showed similar protein structures consisting of four domains: hydrophobic signal peptide domain, cysteine-rich chitin-binding domain, hinge domain, and catalytic domain. The EuNOD-CHT1 gene showed similar expression levels in root nodules and leaves, with no detection of transcripts in the roots. The EuNOD-CHT2 gene was expressed at similarly high levels in the roots and root nodules, but at a very low level in the leaves. In situ hybridization showed that EuNOD-CHT1 transcripts were strongly detected in the meristem zone, but weakly detected in the outer cortex layer of the root nodule and in the uninfected cells of the fixation zone. On the other hand, EuNOD-CHT2 transcripts were strongly detected in the infected cells of the fixation zone and central vascular system, but weakly detected in the senescence zone. Our results suggest that the two chitinases may play different biological roles in the root nodule. EuNOD-CHT2 may be involved in a defense response against internal symbionts, external pathogens, or both, while EuNOD-CHT1 may be involved in normal plant development as well as in a defensive role against external pathogens.

Cloning and expression of mitochondrial MnSOD from the small radish (Raphanus sativus L.).

A cDNA clone for a mitochondrial MnSOD was isolated from a cDNA library derived from seedlings of the small radish (Raphanus sativus L.). The cDNA clone, RsMnSOD, encoded a polypeptide with a predicted molecular mass of 25.4 kDa and calculated pI of 8.77. Its deduced amino acid sequence was 93% homologous with MnSOD of Arabidopsis. RNA gel blot analysis showed that RsMnSOD transcripts were most abundant in leaves, followed by roots and hypocotyls, whereas transcripts of RsFeSOD and RsCu/ZnSOD were not detected in roots. The hypocotyls of germinated seedlings turned green and finally red in response to white light. These color changes were accompanied by increases in RsMnSOD and RsCu/ZnSOD mRNA. In addition, RsMnSOD expression was strongly induced by osmotic stress, moderately induced by phytohormones such as ABA and IAA, and not induced by xenobiotics other than cercosporin.

Cold-inducible transcription factor, CaCBF, is associated with a homeodomain leucine zipper protein in hot pepper (Capsicum annuum L.).

C-Repeat/drought responsive element binding factor (CBF1/DREB1b) is a well known transcriptional activator that is induced at low temperature and in turn induces the CBF regulon (CBF-targeted genes). We have cloned and characterized two CBF1-like cDNAs, CaCBF1A and CaCBF1B, from hot pepper. CaCBF1A and CaCBF1B were not produced in response to mechanical wounding or abscisic acid but were induced by low-temperature stress at 4 degrees . When plants were returned to 25 degrees , their transcript levels of the CBF1-like genes decreased markedly within 40 min. Long-term exposure to chilling resulted in continuous expression of these genes. The critical temperature for induction of CaCBF1A was between 10 and 15 degrees . Low temperature led to its transcription in roots, stems, leaves, fruit without seeds, and apical meristems, and a monoclonal antibody against it revealed a significant increase in CaCBF1A protein by 4 h at 4 degrees . Two-hybrid screening led to the isolation of an homeodomain leucine zipper (HD-Zip) protein that interacts with CaCBF1B. Expression of HD-Zip was elevated by low temperature and drought.

Molecular cloning and complementation analysis of nifV gene from Frankia EuIK1 strain.

The nifV gene from the Frankia EuIK1 strain, a symbiont of Elaeagnus umbellata, was cloned and a complementation test using the Klebsiella pneumoniae nifV mutant was performed to verify its function. The nifV ORF consists of 1245 bp, which encodes 414 amino acids. However, the putative promoter and Shine-Dalgarno sequences were not found in the 5' region of the ORF. The Frankia EuIK1 nifV ORF showed about a 70% nucleotide identity and 80% amino acid similarity with that of Frankia sp. FaC1. In the upstream region of the nifV, a putative ORF that showed a 51% nucleotide identity with the afcD gene from Burkholderia cepacia BC11 was found. The other partial ORF that showed a 59% identity with the pkaD gene from Streptomyces coelicolor A(3) was found in the downstream region. In this respect, Frankia EuIK1 nifV has an unusual location on the genome, considering the nif gene organization. A phylogenetic analysis revealed that the NifV from Frankia EuIK1 was close to those from two Alnus-infective Frankia species, and they were grouped with those of the alpha-class proteobacteria, supporting the vertical descent of nifV. The transcription and function of Frankia EuIK1 nifV were verified by a RT-PCR analysis and complementation test with the K. pneumoniae mutant, respectively. These results suggested that Frankia EuIK1 nifV is a functional gene.

Analysis of the root nodule-enhanced transcriptome in soybean.

For high throughput screening of root nodule-enhanced genes, cDNA libraries specific for three different developmental stages of soybean root nodules were constructed after inoculation with Bradyrhizobium japonicum USDA110. 5,469 cDNA clones were sequenced and grouped into 2,511 non-redundant (nr) ESTs consisting of 769 contigs and 1,742 singletons. Using similarity searches against several public databases we constructed a functional classification of the ESTs into root nodule-specific nodulin genes, stress-responsive genes and genes related to carbon and nitrogen metabolism. We also constructed a cDNA microarray with 382 selected clones that appeared to be up-regulated in the root nodule. Using the microarray we compared the transcript levels of uninfected roots and root nodules from four developmental stages. We identified 81 genes that were differentially expressed, and grouped them into seven clusters according to the similarity of their expression profiles, using a hierarchical clustering algorithm. Clusters 1, 2, 3, and 6, comprised of 58 genes, showed root nodule-enhanced expression. The information from this study will be used to analyze the roles of root nodule-specific genes and signaling pathways during root nodule development.

Arabidopsis thaliana Remorins Interact with SnRK1 and Play a Role in Susceptibility to Beet Curly Top Virus and Beet Severe Curly Top Virus.

Remorins, a family of plant-specific proteins containing a variable N-terminal region and conserved C-terminal domain, play a role in various biotic and abiotic stresses, including host-microbe interactions. However, their functions remain to be completely elucidated, especially for the Arabidopsis thaliana remorin group 4 (AtREM4). To elucidate the role of remorins in Arabidopsis, we first showed that AtREM4s have typical molecular characteristics of the remorins, such as induction by various types of biotic and abiotic stresses, localization in plasma membrane and homo- and hetero-oligomeric interaction. Next, we showed that their loss-of-function mutants displayed reduced susceptibility to geminiviruses, Beet Curly Top Virus and Beet Severe Curly Top Virus, while overexpressors enhanced susceptibility. Moreover, we found that they interacted with SnRK1, which phosphorylated AtREM4.1, and were degraded by the 26S proteasome pathway. These results suggest that AtREM4s may be involved in the SnRK1-mediated signaling pathway and play a role as positive regulators of the cell cycle during geminivirus infection.

Soybean MAPK, GMK1 is dually regulated by phosphatidic acid and hydrogen peroxide and translocated to nucleus during salt stress.

Mitogen-activated protein kinase (MAPK) is activated by various biotic and abiotic stresses. Salt stress induces two well-characterized MAPK activating signaling molecules, phosphatidic acid (PA) via phospholipase D and phospholipase C, and reactive oxygen species (ROS) via nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase. In our previous study, the activity of soybean MAPK, GMK1 was strongly induced within 5 min of 300 mM NaCl treatment and this early activity was regulated by PA. In this study, we focused on the regulation of GMK1 at the later stage of the salt stress, because its activity was strongly persistent for up to 30 min. H(2)O(2) activated GMK1 even in the presence of PA generation inhibitors, but GMK1 activity was greatly decreased in the presence of diphenyleneiodonium, an inhibitor of NADPH-oxidase after 5 min of the treatment. On the contrary, the n-butanol and neomycin reduced GMK1 activity within 5 min of the treatment. Thus, GMK1 activity may be sustained by H(2)O(2) 10 min after the treatment. Further, GMK1 was translocated into the nucleus 60 min after NaCl treatment. In the relationship between GMK1 and ROS generation, ROS generation was reduced by SB202190, a MAPK inhibitor, but was increased in protoplast overexpressing TESD-GMKK1. However, these effects were occurred at prolonged time of NaCl treatment. These data suggest that GMK1 indirectly regulates ROS generation. Taken together, we propose that soybean GMK1 is dually regulated by PA and H(2)O(2) at a time dependant manner and translocated to the nucleus by the salt stress signal.

Comprehensive analysis of AHL homologous genes encoding AT-hook motif nuclear localized protein in rice.

The AT-hook motif is a small DNA-binding protein motif that has been found in the high mobility group of non-histone chromosomal proteins. The Arabidopsis genome contains 29 genes encoding the AT-hook motif DNA-binding protein (AHL). Recent studies of Arabidopsis genes (AtAHLs) have revealed that they might play diverse functional roles during plant growth and development. In this report, we mined 20 AHL genes (OsAHLs) from the rice genome database using AtAHL genes as queries and characterized their molecular features. A phylogenetic tree revealed that OsAHL proteins can be classified into 2 evolutionary clades. Tissue expression pattern analysis revealed that all of the OsAHL genes might be functionally expressed genes with 3 distinct expression patterns. Nuclear localization analysis using transgenic Arabidopsis showed that several OsAHL proteins are exclusively localized in the nucleus, indicating that they may act as architectural transcription factors to regulate expression of their target genes during plant growth and development.

The regulation of DWARF4 expression is likely a critical mechanism in maintaining the homeostasis of bioactive brassinosteroids in Arabidopsis.

Mutants that are defective in brassinosteroid (BR) biosynthesis or signaling display severely retarded growth patterns due to absence of growth-promoting effects by BRs. Arabidopsis (Arabidopsis thaliana) DWARF4 (DWF4) catalyzes a flux-determining step in the BR biosynthetic pathways. Thus, it is hypothesized that the tissues of DWF4 expression may represent the sites of BR biosynthesis in Arabidopsis. Here we show that DWF4 transcripts accumulate in the actively growing tissues, such as root, shoot apices with floral clusters, joint tissues of root and shoot, and dark-grown seedlings. Conforming to the RNA gel-blot analysis, DWF4:beta-glucuronidase (GUS) histochemical analyses more precisely define the tissues that express the DWF4 gene. Examination of the endogenous levels of BRs in six and seven different tissues of wild type and brassinosteroid insensitive1-5 mutant, respectively, revealed that BRs are significantly enriched in roots, shoot tips, and joint tissues of roots and shoots. In addition, DWF4:GUS expression was negatively regulated by BRs. DWF4:GUS activity was increased by treatment with brassinazole, a BR biosynthetic inhibitor, and decreased by exogenous application of bioactive BRs. When DWF4:GUS was expressed in a different genetic background, its level was down-regulated in brassinazole resistant1-D, confirming that BRASSINAZOLE RESISTANT1 acts as a negative regulator of DWF4. Interestingly, in the brassinosteroid insensitive2/dwf12-1D background, DWF4:GUS expression was intensified and delocalized to elongating zones of root, suggesting that BRASSINOSTEROID INSENSITIVE2 is an important factor that limits DWF4 expression. Thus, it is likely that the DWF4 promoter serves as a focal point in maintaining homeostasis of endogenous bioactive BR pools in specific tissues of Arabidopsis.

Arabidopsis cyp51 mutant shows postembryonic seedling lethality associated with lack of membrane integrity.

CYP51 exists in all organisms that synthesize sterols de novo. Plant CYP51 encodes an obtusifoliol 14alpha-demethylase involved in the postsqualene sterol biosynthetic pathway. According to the current gene annotation, the Arabidopsis (Arabidopsis thaliana) genome contains two putative CYP51 genes, CYP51A1 and CYP51A2. Our studies revealed that CYP51A1 should be considered an expressed pseudogene. To study the functional importance of the CYP51A2 gene in plant growth and development, we isolated T-DNA knockout alleles for CYP51A2. Loss-of-function mutants for CYP51A2 showed multiple defects, such as stunted hypocotyls, short roots, reduced cell elongation, and seedling lethality. In contrast to other sterol mutants, such as fk/hydra2 and hydra1, the cyp51A2 mutant has only minor defects in early embryogenesis. Measurements of endogenous sterol levels in the cyp51A2 mutant revealed that it accumulates obtusifoliol, the substrate of CYP51, and a high proportion of 14alpha-methyl-delta8-sterols, at the expense of campesterol and sitosterol. The cyp51A2 mutants have defects in membrane integrity and hypocotyl elongation. The defect in hypocotyl elongation was not rescued by the exogenous application of brassinolide, although the brassinosteroid-signaling cascade is apparently not affected in the mutants. Developmental defects in the cyp51A2 mutant were completely rescued by the ectopic expression of CYP51A2. Taken together, our results demonstrate that the Arabidopsis CYP51A2 gene encodes a functional obtusifoliol 14alpha-demethylase enzyme and plays an essential role in controlling plant growth and development by a sterol-specific pathway.

Leptin induces apoptosis via ERK/cPLA2/cytochrome c pathway in human bone marrow stromal cells.

Leptin, the Ob gene product, has emerged recently as a key regulator of bone mass. However, the mechanism mediating leptin effect remains controversial. Because the action of leptin is dependent on its receptors, we analyzed their expression in osteoblast-lineage primary human bone marrow stromal cells (hBMSC). Both the short and long forms of leptin receptors were detected in hBMSC. Leptin significantly decreased the viability of hBMSC. This cytotoxic effect was prevented by Z-Val-Ala-Asp-fluoromethylketone, a pan-caspase inhibitor, implicating that leptin-induced hBMSC death was caspase-dependent. Further investigation demonstrated that leptin activated caspase-3 and caspase-9, but not caspase-8, and increased the cleavage of poly-(ADP-ribose) polymerase and cytochrome c release into cytosol. Leptin activated ERK, but not p38 and JNK, and up-regulated cPLA2 activity; the latter was abolished by pre-treatment of cells with the MEK inhibitor (PD98059 or U0126) or cPLA2 inhibitor (AACOCF3). PD98059, U0126, and AACOCF3 also diminished the leptin-induced cytochrome c release into cytosol, cell death, and caspase-3 activation. These data indicated that leptin induced hBMSC apoptosis via ERK/cPLA2/cytochrome c pathway with activation of caspase-9 and caspase-3, and cleavage of poly(ADP-ribose) polymerase. To our knowledge, this is the first study demonstrating the direct detrimental effect of leptin on bone cells.