Elisheva Smith

Bone Morphogenetic Protein‐2 Restores Mineralization in Glucocorticoid‐Inhibited MC3T3‐E1 Osteoblast Cultures

The anti‐glucocorticoid potential of BMP‐2 in osteoblasts was tested in MC3T3‐E1 cells using dexamethasone (1 μM) and rhBMP‐2 (10 or 100 ng/ml). rhBMP‐2 restored mineralization but not condensation or collagen accumulation. These results demonstrate the potential and limitations of BMPs in counteracting glucocorticoids.

Brief bone morphogenetic protein 2 treatment of glucocorticoid-inhibited MC3T3-E1 osteoblasts rescues commitment-associated cell cycle and mineralization without alteration of Runx2.

Glucocorticoids (GCs) inhibit bone formation in vivo. In MC3T3-E1 osteoblasts, chronic administration of 1 μm dexamethasone (DEX) starting at confluency results in >98% inhibition of bone morphogenetic protein 2 (BMP-2) expression and apatite mineral deposition. Here, it is shown that brief exposure to recombinant human BMP-2 (rhBMP-2), as short as 6 h, is sufficient to induce irreversible commitment to mineralization in DEX-treated cultures. RhBMP-2 dose dependently rescued mineralization but not collagen accumulation in DEX-inhibited cultures. The selective restoration of mineralization was evident in the higher mineral to matrix ratios of DEX/rhBMP-2 co-treated cultures compared with control. We tested the involvement of the runt-related transcription factor 2 (Runx2) in the DEX inhibition and rhBMP-2 rescue of mineralization. Surprisingly, DEX did not decrease Runx2 DNA binding activity, transactivation, or association with the endogenous osteocalcin gene promoter. Furthermore, the rhBMP-2 rescue did not involve Runx2 stimulation, suggesting an important role for factors other than Runx2 in BMP-2 action. Finally, we studied the differentiation-related cell cycle, which persists during commitment to mineralization in untreated cultures, but is inhibited along with mineralization in DEX-treated cultures. Although both rhBMP-2 alone and DEX alone were antimitogenic, rhBMP-2 stimulated this cell cycle in DEX-inhibited cultures. In conclusion, brief rhBMP-2 treatment restores mineralization in DEX-inhibited MC3T3-E1 osteoblasts via a mechanism different from Runx2 stimulation. This restoration may be functionally related to the accompanying rescue of the differentiation-related cell cycle. The efficacy of short term BMP-2 treatment supports the potential of short-lived BMP vectors for skeletal therapy in both traditional and gene therapeutic approaches.

Lef1 Haploinsufficient Mice Display a Low Turnover and Low Bone Mass Phenotype in a Gender- and Age-Specific Manner

We investigated the role of Lef1, one of the four transcription factors that transmit Wnt signaling to the genome, in the regulation of bone mass. Microcomputed tomographic analysis of 13- and 17-week-old mice revealed significantly reduced trabecular bone mass in Lef1+/− females compared to littermate wild-type females. This was attributable to decreased osteoblast activity and bone formation as indicated by histomorphometric analysis of bone remodeling. In contrast to females, bone mass was unaffected by Lef1 haploinsufficiency in males. Similarly, females were substantially more responsive than males to haploinsufficiency in Gsk3β, a negative regulator of the Wnt pathway, displaying in this case a high bone mass phenotype. Lef1 haploinsufficiency also led to low bone mass in males lacking functional androgen receptor (AR) (tfm mutants). The protective skeletal effect of AR against Wnt-related low bone mass is not necessarily a result of direct interaction between the AR and Wnt signaling pathways, because Lef1+/− female mice had normal bone mass at the age of 34 weeks. Thus, our results indicate an age- and gender-dependent role for Lef1 in regulating bone formation and bone mass in vivo. The resistance to Lef1 haploinsufficiency in males with active AR and in old females could be due to the reduced bone turnover in these mice.

Biosynthesis of Osteogenic Growth Peptide via Alternative Translational Initiation at AUG85 of Histone H4 mRNA

The osteogenic growth peptide (OGP) is an extracellular mitogen identical to the histone H4 (H4) COOH-terminal residues 90–103, which regulates osteogenesis and hematopoiesis. By Northern analysis, OGP mRNA is indistinguishable from H4 mRNA. Indeed, cells transfected with a construct encoding [His102]H4 secreted the corresponding [His13]OGP. These results suggest production of OGP from H4 genes. Cells transfected with H4-chloramphenicol acetyltransferase (CAT) fusion genes expressed both “long” and “short” CAT proteins. The short CAT was retained following an ATG → TTG mutation of the H4 ATG initiation codon, but not following mutation of the in-frame internal ATG85 codon, which, unlike ATG1, resides within a perfect context for translational initiation. These results suggest that a PreOGP is translated starting at AUG85. The translational initiation at AUG85could be inhibited by optimizing the nucleotide sequence surrounding ATG1 to maximally support upstream translational initiation, thus implicating leaky ribosomal scanning in usage of the internal AUG. Conversion of the predicted PreOGP to OGP was shown in a cell lysate system using synthetic [His102]H4-(85–103) as substrate. Together, our results demonstrate that H4 gene expression diverges at the translational level into the simultaneous parallel production of both H4, a nuclear structural protein, and OGP, an extracellular regulatory peptide.

Leaky ribosomal scanning in mammalian genomes: significance of histone H4 alternative translation in vivo

Like alternative splicing, leaky ribosomal scanning (LRS), which occurs at suboptimal translational initiation codons, increases the physiological flexibility of the genome by allowing alternative translation. Comprehensive analysis of 22 208 human mRNAs indicates that, although the most important positions relative to the first nucleotide of the initiation codon, −3 and +4, are usually such that support initiation (A−3 = 42%, G−3 = 36% and G+4 = 47%), only 37.4% of the genes adhere to the purine (R)−3/G+4 rule at both positions simultaneously, suggesting that LRS may occur in some of the remaining (62.6%) genes. Moreover, 12.5% of the genes lack both R−3 and G+4, potentially leading to sLRS. Compared with 11 genes known to undergo LRS, 10 genes with experimental evidence for high fidelity A+1T+2G+3 initiation codons adhered much more strongly to the R−3/G+4 rule. Among the intron-less histone genes, only the H3 genes adhere to the R−3/G+4 rule, while the H1, H2A, H2B and H4 genes usually lack either R−3 or G+4. To address in vivo the significance of the previously described LRS of H4 mRNAs, which results in alternative translation of the osteogenic growth peptide, transgenic mice were engineered that ubiquitously and constitutively express a mutant H4 mRNA with an A+1→T+1 mutation. These transgenic mice, in particular the females, have a high bone mass phenotype, attributable to increased bone formation. These data suggest that many genes may fulfill cryptic functions by LRS.

Opposing effects of glucocorticoids and Wnt signaling on Krox20 and mineral deposition in osteoblast cultures.

Krox20 is expressed in osteoblasts and chondrocytes, and is required for trabecular bone formation during embryogenesis. Here we show by RT‐qPCR and Western blot analysis that Krox20 is up‐regulated during late stages of osteoblast differentiation in culture. Glucocorticoids (GCs) rapidly inhibit the expression of Krox20 as well its co‐activator, HCF‐1, resulting in inhibition of the Osteocalcin Krox20‐binding Enhancer (OKE). GCs also inhibit expression of EGR1, EGR3, and EGR4. OKE activity, which is dependent on the presence of Runx2, was independent of the osteocalcin promoter Runx2 binding site. In contrast to GCs, activation of the Wnt, but not the BMP or the PTH signaling pathways, stimulated Krox20 expression as well as activity of the OKE. GC‐mediated suppression of Krox20 expression was compromised, albeit not completely, in the presence of DKK1, suggesting that the inhibition occurs in both Wnt‐dependent and Wnt‐independent manners. Furthermore, Wnt3A partially rescued Krox20 expression in GC‐arrested osteoblast cultures and this was accompanied by rescue of mineralization. These findings are consistent with a role for Krox20 in osteoblast function and suggest that this transcription factor may contribute to the opposing effects of GCs and Wnt signaling on bone formation. J. Cell. Biochem. 103: 1938–1951, 2007.

Post-proliferative cyclin E-associated kinase activity in differentiated osteoblasts: inhibition by proliferating osteoblasts and osteosarcoma cells.

Spontaneous differentiation of normal diploid osteoblasts in culture is accompanied by increased cyclin E associated kinase activity on (1) the retinoblastoma susceptibility protein pRB, (2) the p107 RB related protein, and (3) two endogenous cyclin E‐associated substrates of 78 and 105 kD. Activity of the differentiation‐related cyclin E complexes (diff.ECx) is not recovered in cdc2 or cdk2 immunoprecipitates. Phosphorylation of both the 105 kD endogenous substrate and the p107 exogenous substrate is sensitive to inhibitory activity (diff.ECx‐i) present in proliferating osteoblasts. This inhibitory activity is readily recruited by the cyclin E complexes of differentiated osteoblasts but is not found in cyclin E immunoprecipitates of the proliferating cells themselves. Strong inhibitory activity on diff.ECx kinase activity is excerted by proliferating ROS 17/2.8 osteosarcoma cells. However, unlike the normal diploid cells, the diff.ECx‐i activity of proliferating ROS 17/2.8 cells is recovered by cyclin E immunoprecipitation. The cyclin‐dependent kinase inhibitor p21CIP1/WAF1 inhibits diff.ECx kinase activity. Thus, our results suggest the existence of a unique regulatory system, possibly involving p21CIP1/WAF1, in which inhibitory activity residing in proliferating cells is preferentially targeted towards differentiation‐related cyclin E‐associated kinase activity. J. Cell. Biochem. 66:141‐152, 1997.

The honeybee Apis mellifera contributes to Cucumber green mottle mosaic virus spread via pollination

The tobamovirus Cucumber green mottle mosaic virus (CGMMV) is efficiently transmitted between plants by mechanical contact. So far, no clear evidence has been reported regarding the transmission potential of the virus by beneficial pollinator insects. This study examined the capability of the well-known pollinator honeybee Apis mellifera to transmit CGMMV in cucurbits using melon and cucumber plants as a model. In order to provide a clear answer to that question, five experiments were designed on various scales performed under three environmental conditions. The results show that under protected cropping conditions, CGMMV is transmitted by the honeybees. The location of the beehive in relation to both the CGMMV primary inoculum source and the healthy plants during honeybee foraging plays an important role in the efficiency of CGMMV spread. Furthermore, in the presence of early stage CGMMV-inoculated plants, the efficiency of CGMMV spread to uninoculated plants placed on the honeybees’ path to the beehive may increase. To the authors’ knowledge, CGMMV transmission by honeybees has not yet been shown, and this study can be adopted for other tobamovirus related research.

A New Israeli Tobamovirus Isolate Infects Tomato Plants Harboring Tm-22 Resistance Genes

An outbreak of a new disease infecting tomatoes occurred in October-November 2014 at the Ohad village in Southern Israel. Symptomatic plants showed a mosaic pattern on leaves accompanied occasionally by narrowing of leaves and yellow spotted fruit. The disease spread mechanically and rapidly reminiscent of tobamovirus infection. Epidemiological studies showed the spread of the disease in various growing areas, in the South and towards the Southeast and Northern parts of the country within a year. Transmission electron microscope (TEM) analysis showed a single rod-like form characteristic to the Tobamovirus genus. We confirmed Koch’s postulates for the disease followed by partial host range determination and revealed that tomato cultivars certified to harbor the Tm-22 resistance gene are susceptible to the new viral disease. We further characterized the viral source of the disease using a range of antisera for serological detection and analyzed various virus genera and families for cross-reactivity with the virus. In addition, next generation sequencing of total small RNA was performed on two cultivars grown in two different locations. In samples collected from commercial cultivars across Israel, we found a single virus that caused the disease. The complete genome sequence of the new Israeli tobamovirus showed high sequence identity to the Jordanian isolate of tomato brown rugose fruit virus.

New weed hosts for Cucumber green mottle mosaic virus in wild Mediterranean vegetation

The Tobamovirus Cucumber green mottle mosaic virus (CGMMV) is considered a major risk for cucurbit crop production. In 2013, an outbreak of CGMMV in watermelon fields occurred in Israel causing severe crop damage. This event raised concern regarding the virus preservation potential in weeds within and surrounding cultivated cucurbit crops. Out of 11 surveyed weed species, six were ELISA-positive for CGMMV, although they were asymptomatic: Moluccella laevis, Withania somnifera, Amaranthus graecizans, A. muricatus, Ecballium elaterium and Chrozophora tinctoria. The presence of CGMMV in the weed species was verified by RT-PCR analysis and by bioassay to healthy melon, cucumber and Nicotiana benthamiana plants and to non-infected weed species. These results add species inside the Solanaceae, Euphorbiaceae and Lamiaceae families to CGMMV host range in weeds. Thus, weeds reservoir within and surrounding infected fields needs to be considered as a potential source for CGMMV infection of cultivated plants.