Luigi Moro

Effects of 1- and 6-month spaceflight on bone mass and biochemistry in two humans

The bone mineral density and the biochemical parameters exploring bone cell activities were analyzed in two cosmonauts who spent 1 and 6 months, respectively, in the Russian MIR station. Measurements were performed before the flight, after the flight, and after a recovery period. At the end of the first month, peripheral QCT measurements indicated a slight decrease of trabecular bone mass in the distal tibial metaphysis. However, after 6 months of spaceflight, a more marked loss of trabecular and cortical bones was observed in the tibia, and was still significant after 6 month recovery in the trabecular compartment, whereas a decrease was no longer observed in the cortical envelope. No change was observed in either compartment of the distal radius at any time. Ultrasound BUA of the calcaneus was greatly reduced by the first month, followed by a more dramatic decrease after month 6. Ultrasound SOS detected no change. Parameters reflecting bone formation activity appeared to be depressed after both missions. In contrast, no dramatic change in resorption parameters was observed, except for a trend toward an increase in pyridinoline. In conclusion, the lower weight-bearing bones appeared more sensitive than the upper ones in terms of spaceflight-induced bone loss. This probably explained the absence of marked systemic biochemical data changes. This study further suggests that recovery in the tibial trabecular compartment 6 months after landing was not completed after a 6 month mission.

Chemical and structural characterization of the mineral phase from cortical and trabecular bone

X-ray diffraction, infrared spectroscopy and chemical investigations have been carried out on the inorganic phases from rat cortical and trabecular bone. Although both inorganic phases consist of poorly crystalline B carbonated apatite, several significant differences have been observed. In particular, trabecular bone apatite displays reduced crystallite sizes, Ca/P molar ratio, and carbonate content, and exhibits a greater extent of thermal conversion into beta-tricalcium phosphate than cortical bone apatite. These differences can be related to the different extents of collagen posttranslational modifications exhibited by the two types of bone, in agreement with their different biological functions.

Modifications of bone and connective tissue after orthostatic bedrest

Abstract: Eight male volunteers were submitted to a 6-week anti-orthostatic bedrest trial followed by a 1-month reambulation period. We prospectively monitored whole-body composition by dual-energy X-ray absorptiometry, bone and connective tissue metabolism by biochemical markers and calcium regulating hormones by 1–84 parathyroid hormone and 1,25-dihydroxyvitamin D3. Bone mineral density (BMD) did not vary significantly; however, a trend toward an increase in head BMD and a decrease in trunk, lumbar vertebrae and lower limb BMD was observed. A decrease in the lower limb lean content occurred by day 27 and was maximum by day 42 after the beginning of bedrest; it normalized by day 30 after bedrest. The serum levels of both osteocalcin and C-terminal crosslinked telopeptide of type I collagen increased as a consequence of bedrest. A slight increase in the serum levels of the N-terminal propeptide of type III collagen, a marker of connective tissue metabolism, was observed during the bedrest period. Except for the C-terminal extension propeptide of type I collagen, all markers decreased to baseline pre-immobilization levels during the 1-month recovery phase. Serum PTH and 1,25-dihydroxyvitamin D3 levels were low during the bedrest period and rose during the reambulation phase. These results seem to reflect early changes in bone and connective tissue metabolism as a result of bedrest unloading, but their order of magnitude remains moderate, thus emphasizing the necessity to perform longer-duration trials.

Urinary β-1-galactosyl-0-hydroxylysine (GH) as a marker of collagen turnover of bone

Summaryβ-1-galactosyl-0-hydroxylysine (GH) was measured in the urine of 59 women and 48 men, aged 30–79 years, by High Performance Liquid Chromatography (HPLC) of the dansylated derivative. Vertebral mineral density, measured by quantitative computed tomography (QCT), and urinary GH were inversely correlated (r=−0.74;P<0.001). High rate of bone mineral loss is associated with a high urinary GH excretion. Measurement of GH in urine provides a simple and noninvasive method for the evaluation of the extent of bone resorption in large groups of subjects and appears to be more specific than urinary hydroxyproline excretion.

Alkaline phosphatase binds to collagen; a hypothesis on the mechanism of extravesicular mineralization in epiphyseal cartilage

Affinity chromatography on Sepharose 4B-collagen gels was used to test the affinity of alkaline phosphatase for collagen. Results indicate that 1) alkaline phosphatase of preosseous cartilage binds to collagen probably by electrostatic interactions, 2) this interaction is inhibited by proteoglycan subunits. These results suggest that, in vivo, the formation of a collagen-alkaline phosphatase complex may be a step of the process leading to cartilage calcification.

Extracellular ATP stimulates the early growth response protein 1 (Egr-1) via a protein kinase C-dependent pathway in the human osteoblastic HOBIT cell line.

Extracellular nucleotides exert an important role in controlling cell physiology by activating intracellular signalling cascades. Osteoblast HOBIT cells express P2Y(1) and P2Y(2) G-protein-coupled receptors, and respond to extracellular ATP by increasing cytosolic calcium concentrations. Early growth response protein 1 (Egr-1) is a C(2)H(2)-zinc-finger-containing transcriptional regulator responsible for the activation of several genes involved in the control of cell proliferation and apoptosis, and is thought to have a central role in osteoblast biology. We show that ATP treatment of HOBIT cells increases Egr-1 protein levels and binding activity via a mechanism involving a Ca(2+)-independent protein kinase C isoform. Moreover, hypotonic stress and increased medium turbulence, by inducing ATP release, result in a similar effect on Egr-1. Increased levels of Egr-1 protein expression and activity are achieved at very early times after stimulation (5 min), possibly accounting for a rapid way for changing the osteoblast gene-expression profile. A target gene for Egr-1 that is fundamental in osteoblast physiology, COL1A2, is up-regulated by ATP stimulation of HOBIT cells in a timescale that is compatible with that of Egr-1 activation.

Cross-regulation between Egr-1 and APE/Ref-1 during early response to oxidative stress in the human osteoblastic HOBIT cell line: Evidence for an autoregulatory loop

The Early Growth Response protein (Egr-1) is a C2H2–zinc finger-containing transcriptional regulator involved in the control of cell proliferation and apoptosis. Its DNA-binding activity is redox regulated in vitro through the oxidation–reduction of Cys residues within its DNA-binding domain. APE/Ref-1 is a DNA-repair enzyme with redox modulating activities on several transcription factors. In this study, by evaluating the effects of different stimuli, we found a similar timing of activation being suggestive for a common and co-linear regulation for the two proteins. Indeed, we show that APE/Ref-1 increases the Egr-1 DNA-binding activity in unstimulated osteoblastic HOBIT cells. H2O2 stimulation induces a strong interaction between Egr-1 and APE/Ref-1 at early times upon activation, as assayed by immunoprecipitation experiments. By using a cell transfection approach, we demonstrated the functional role of this interaction showing that two specific Egr-1 target genes, the PTEN phosphatase and the thymidine kinase (TK) genes promoters, are activated by contransfection of APE/Ref-1. Interestingly, by using a cell transfection approach and Chromatin immunoprecipitation assays, we were able to demonstrate that Egr-1 stimulates the transcriptional activity of APE/Ref-1 gene promoter by a direct interaction with specific DNA-binding site on its promoter. Taken together, our data delineate a new molecular mechanism of Egr-1 activation occurring soon after H2O2 stimulation in osteoblastic cells and suggest a model for a positive loop between APE/Ref-1 and Egr-1 that could explain the early transcriptional activation of APE/Ref-1 gene expression.

Collagen type I of rat cortical and trabecular bone differs in the extent of posttranslational modifications

This study sought to evaluate whether the architecture of the matrix of cortical and trabecular bone is exactly the same. For this purpose we analyzed the extent of some posttranslational modifications of type I collagen, which is the major component of bone matrix. Ten female and 10 male 100-day-old rats were sacrificed and the content of hydroxylysine, glycosylated hydroxylysine, and pyridinium cross-links of collagen from cortical and trabecular bone was determined. The amount of each compound was expressed as a molar ratio with hydroxyproline. The collagen posttranslational modification pattern appears to be the same in both sexes but with a higher extent of differences in females compared with males. Comparing cortical and trabecular bone, the former contains a higher amount of hydroxylysine residues whereas in the latter, glycosylation of hydroxylysine is higher and pyridinium cross-link concentration is lower. Moreover, an inverse linear relationship between glycosylated hydroxylysine and pyridinium crosslinks concentration was established, both for female (r=−0.455,P=0.04) and male rats (r=−0.426;P=0.06). This paper discusses what these findings may mean in functional terms.

Identification of secondary targets of N-containing bisphosphonates in mammalian cells via parallel competition analysis of the barcoded yeast deletion collection

BackgroundNitrogen-containing bisphosphonates are the elected drugs for the treatment of diseases in which excessive bone resorption occurs, for example, osteoporosis and cancer-induced bone diseases. The only known target of nitrogen-containing bisphosphonates is farnesyl pyrophosphate synthase, which ensures prenylation of prosurvival proteins, such as Ras. However, it is likely that the action of nitrogen-containing bisphosphonates involves additional unknown mechanisms. To identify novel targets of nitrogen-containing bisphosphonates, we used a genome-wide high-throughput screening in which 5,936 Saccharomyces cerevisiae heterozygote barcoded mutants were grown competitively in the presence of sub-lethal doses of three nitrogen-containing bisphosphonates (risedronate, alendronate and ibandronate). Strains carrying deletions in genes encoding potential drug targets show a variation of the intensity of their corresponding barcodes on the hybridization array over the time.ResultsWith this approach, we identified novel targets of nitrogen-containing bisphosphonates, such as tubulin cofactor B and ASK/DBF4 (Activator of S-phase kinase). The up-regulation of tubulin cofactor B may explain some previously unknown effects of nitrogen-containing bisphosphonates on microtubule dynamics and organization. As nitrogen-containing bisphosphonates induce extensive DNA damage, we also document the role of DBF4 as a key player in nitrogen-containing bisphosphonate-induced cytotoxicity, thus explaining the effects on the cell-cycle.ConclusionsThe dataset obtained from the yeast screen was validated in a mammalian system, allowing the discovery of new biological processes involved in the cellular response to nitrogen-containing bisphosphonates and opening up opportunities for development of new anticancer drugs.

Posttranslational modifications of bone collagen type I are related to the function of rat femoral regions.

Abstract. This study analyzes the relationship between the function of femoral regions in the rat and the extent of collagen type I posttranslational modifications, to assess whether the different functional roles, i.e., mechanical or metabolic, of the bone tissues are related to the molecular structure of the matrix. For this purpose, 18 female, 100-day-old Sprague-Dawley rats were sacrificed, under anesthesia, and their femurs were removed and dissected free of adhering tissue. The spongy bone of the proximal metaphysis and the diaphysis were then selected as regions exerting prevalently a mechanical function, and the spongy bone of the distal metaphysis was selected as mainly related to metabolic function. Bone prepared from these regions was used to extract and purify the major component of the matrix, type I collagen. The content of hydroxyproline, hydroxylysine, glycosylated hydroxylysine, and pyridinium crosslinks was evaluated and the amount of each compound was expressed as a molar ratio to hydroxyproline. The amount of glycosylated hydroxylysine and pyridinium crosslinks in the distal metaphysis are significantly different from the amounts measured both in the diaphysis and the proximal metaphysis. On the contrary, the amounts of the same compounds in the diaphysis and the proximal metaphysis are statistically the same. The amount of free hydroxylysine, however, appears to be different in the proximal metaphysis and in the diaphysis. The conclusion is that matrix composition differs among different skeletal regions according to the main function they exert.