Functional coupling between the extracellular matrix and nuclear lamina by Wnt signaling in progeria.

The segmental premature aging disease Hutchinson-Gilford Progeria (HGPS) is caused by a truncated and farnesylated form of Lamin A. In a mouse model for HGPS, a similar Lamin A variant causes the proliferative arrest and death of postnatal, but not embryonic, fibroblasts. Arrest is due to an inability to produce a functional extracellular matrix (ECM), because growth on normal ECM rescues proliferation. The defects are associated with inhibition of canonical Wnt signaling, due to reduced nuclear localization and transcriptional activity of Lef1, but not Tcf4, in both mouse and human progeric cells. Defective Wnt signaling, affecting ECM synthesis, may be critical to the etiology of HGPS because mice exhibit skeletal defects and apoptosis in major blood vessels proximal to the heart. These results establish a functional link between the nuclear envelope/lamina and the cell surface/ECM and may provide insights into the role of Wnt signaling and the ECM in aging.

Association between low lean body mass and osteoporotic fractures after menopause.

OBJECTIVE: This study evaluated dual-energy x-ray absorptiometry-assessed whole-body bone-muscle relationship (bone mineral content/lean mass [BMC/LM]) as an indicator of its nonmechanical perturbations (ie, systemic) in pre- and postmenopausal women. A total of 3,205 women were studied, either healthy (no fracture [No Fx] groups, 1,035 premenopausal, 1,556 postmenopausal) or with recent fractures (Fx groups, 139 premenopausal, 475 postmenopausal) located at osteoporotic sites (hip, spine, long-bone metaphyses; Type II Fx, n = 386) or at other skeletal sites (Type I Fx, n = 228) to evaluate the impact of decreased muscle mass on fracture incidence before and after menopause. DESIGN: SD-scored graphs of BMC/LM proportionality were obtained from the No Fx groups as normal references. Based on the reference BMC versus LM curves obtained from their respective No Fx pre- and postmenopausal controls, BMC-LM SD scores were calculated for all women with fractures. RESULTS: BMC-LM SD scores in all premenopausal women with fractures and in Type I Fx postmenopausal women were similar to the reference. In contrast, SD scores in Type II Fx postmenopausal women were lower than the reference, especially in those with hip fractures. Except for Type II Fx postmenopausal women, all groups showed linear and similar BMC versus LM curves. Type II Fx postmenopausal women showed nonlinear relationships, with progressively decreasing BMC and BMC-LM SD scores as their LM decreased. CONCLUSIONS: Results suggest that both LM and BMC-LM SD scores can help to differentiate between systemic and mechanical (disuse-related) osteopenia/osteoporosis after menopause. Low LM values or BMC-LM SD scores seem to constitute additional fracture risk factors beyond those usually detected in premenopausal women or in women with other types of fractures. This application of dual-energy x-ray absorptiometry technology may lead to more effective diagnosis and treatment at low cost.

Prelamin A farnesylation and progeroid syndromes.

Hutchinson-Gilford progeria syndrome (HGPS) is caused by a LMNA mutation that leads to the synthesis of a mutant prelamin A that is farnesylated but cannot be further processed to mature lamin A. A more severe progeroid disorder, restrictive dermopathy (RD), is caused by the loss of the prelamin A-processing enzyme, ZMPSTE24. The absence of ZMPSTE24 prevents the endoproteolytic processing of farnesyl-prelamin A to mature lamin A and leads to the accumulation of farnesyl-prelamin A. In both HGPS and RD, the farnesyl-prelamin A is targeted to the nuclear envelope, where it interferes with the integrity of the nuclear envelope and causes misshapen cell nuclei. Recent studies have shown that the frequency of misshapen nuclei can be reduced by treating cells with a farnesyltransferase inhibitor (FTI). Also, administering an FTI to mouse models of HGPS and RD ameliorates the phenotypes of progeria. These studies have prompted interest in testing the efficacy of FTIs in children with HGPS.

Protein farnesyltransferase inhibitors and progeria.

Genetic mutations that lead to an accumulation of farnesyl-prelamin A cause progeroid syndromes, including Hutchinson-Gilford progeria syndrome. It seemed possible that the farnesylated form of prelamin A might be toxic to mammalian cells, accounting for all the disease phenotypes that are characteristic of progeria. This concept led to the hypothesis that protein farnesyltransferase inhibitors (FTIs) might ameliorate the disease phenotypes of progeria in mouse models. Thus far, two different mouse models of progeria have been examined. In both models, FTIs improved progeria-like disease phenotypes. Here, prelamin A post-translational processing is discussed and several mutations underlying human progeroid syndromes are described. In addition, recent data showing that FTIs ameliorate disease phenotypes in a pair of mouse models of progeria are discussed.

A farnesyltransferase inhibitor improves disease phenotypes in mice with a Hutchinson-Gilford progeria syndrome mutation.

Hutchinson-Gilford progeria syndrome (HGPS) is caused by the production of a truncated prelamin A, called progerin, which is farnesylated at its carboxyl terminus. Progerin is targeted to the nuclear envelope and causes misshapen nuclei. Protein farnesyltransferase inhibitors (FTI) mislocalize progerin away from the nuclear envelope and reduce the frequency of misshapen nuclei. To determine whether an FTI would ameliorate disease phenotypes in vivo, we created gene-targeted mice with an HGPS mutation (LmnaHG/+) and then examined the effect of an FTI on disease phenotypes. LmnaHG/+ mice exhibited phenotypes similar to those in human HGPS patients, including retarded growth, reduced amounts of adipose tissue, micrognathia, osteoporosis, and osteolytic lesions in bone. Osteolytic lesions in the ribs led to spontaneous bone fractures. Treatment with an FTI increased adipose tissue mass, improved body weight curves, reduced the number of rib fractures, and improved bone mineralization and bone cortical thickness. These studies suggest that FTIs could be useful for treating humans with HGPS.

Prelamin A and lamin A appear to be dispensable in the nuclear lamina.

Lamin A and lamin C, both products of Lmna, are key components of the nuclear lamina. In the mouse, a deficiency in both lamin A and lamin C leads to slow growth, muscle weakness, and death by 6 weeks of age. Fibroblasts deficient in lamins A and C contain misshapen and structurally weakened nuclei, and emerin is mislocalized away from the nuclear envelope. The physiologic rationale for the existence of the 2 different Lmna products lamin A and lamin C is unclear, although several reports have suggested that lamin A may have particularly important functions, for example in the targeting of emerin and lamin C to the nuclear envelope. Here we report the development of lamin C-only mice (Lmna(LCO/LCO)), which produce lamin C but no lamin A or prelamin A (the precursor to lamin A). Lmna(LCO/LCO) mice were entirely healthy, and Lmna(LCO/LCO) cells displayed normal emerin targeting and exhibited only very minimal alterations in nuclear shape and nuclear deformability. Thus, at least in the mouse, prelamin A and lamin A appear to be dispensable. Nevertheless, an accumulation of farnesyl-prelamin A (as occurs with a deficiency in the prelamin A processing enzyme Zmpste24) caused dramatically misshapen nuclei and progeria-like disease phenotypes. The apparent dispensability of prelamin A suggested that lamin A-related progeroid syndromes might be treated with impunity by reducing prelamin A synthesis. Remarkably, the presence of a single Lmna(LCO) allele eliminated the nuclear shape abnormalities and progeria-like disease phenotypes in Zmpste24-/- mice. Moreover, treating Zmpste24-/- cells with a prelamin A-specific antisense oligonucleotide reduced prelamin A levels and significantly reduced the frequency of misshapen nuclei. These studies suggest a new therapeutic strategy for treating progeria and other lamin A diseases.

A protein farnesyltransferase inhibitor ameliorates disease in a mouse model of progeria.

Progerias are rare genetic diseases characterized by premature aging. Several progeroid disorders are caused by mutations that lead to the accumulation of a lipid-modified (farnesylated) form of prelamin A, a protein that contributes to the structural scaffolding for the cell nucleus. In progeria, the accumulation of farnesyl-prelamin A disrupts this scaffolding, leading to misshapen nuclei. Previous studies have shown that farnesyltransferase inhibitors (FTIs) reverse this cellular abnormality. We tested the efficacy of an FTI (ABT-100) in Zmpste24-deficient mice, a mouse model of progeria. The FTI-treated mice exhibited improved body weight, grip strength, bone integrity, and percent survival at 20 weeks of age. These results suggest that FTIs may have beneficial effects in humans with progeria.

Reference charts for the relationships between dual-energy X-ray absorptiometry-assessed bone mineral content and lean mass in 3,063 healthy men and premenopausal and postmenopausal women.

Correlations between dual-energy X-ray absorptiometry (DXA)-assessed bone mineral content and lean mass (BMC-LM curves), and between BMC/LM ratio and age ([BMC/LM]-age curves), were analyzed in the whole body (WB), the upper limbs (ULs) and the lower limbs (LLs) of 3,063 healthy Hispanic adults. Groups of 472 men aged 25-87 years, 1,035 premenopausal (pre-MP) women aged 27-54 years, and 1,556 post-menopausal (post-MP) women aged 48-93 years were studied with a GE-Lunar DPX-Plus device. BMC-LM curves confirmed previous observations that BMC and LM masses always correlate linearly, with similar slopes within each region, but differing in intercepts according to gender and hormonal status. Multiple regression tests showed little or no independent interaction of body weight or height with those relationships. [BMC/LM]-age curves were flat in men but showed the positive influence of estrogens throughout the age range in women. Z-scored graphs of all the corresponding relationships were compiled, showing the confidence intervals for means +/-1, +/-2, and +/-3 SDs of the data (+/-1, +/-2, +/-3 z-scores) along BMC-LM and [BMC/LM]-age curves. These charts are proposed as references for assessing how well bone mass (as assessed by BMC) and muscle mass (assumed proportional to LM) follow the natural anthropometric/biomechanical proportionality in Hispanic men and women within the age range studied, employing similar devices. Charts for LLs, showing the lowest variance amongst the studied correlations and approaching the origin as an exclusive feature, could provide the most accurate reference curves. Differences between data from ULs and LLs may provide information about any eventual interaction of body-weight bearing with the general results. The proposed analysis may provide useful information for approaching a differential diagnosis between disuse-related and other types of osteopenias employing only DXA.

Blocking protein farnesyltransferase improves nuclear blebbing in mouse fibroblasts with a targeted Hutchinson-Gilford progeria syndrome mutation.

Hutchinson-Gilford progeria syndrome (HGPS), a progeroid syndrome in children, is caused by mutations in LMNA (the gene for prelamin A and lamin C) that result in the deletion of 50 aa within prelamin A. In normal cells, prelamin A is a "CAAX protein" that is farnesylated and then processed further to generate mature lamin A, which is a structural protein of the nuclear lamina. The mutant prelamin A in HGPS, which is commonly called progerin, retains the CAAX motif that triggers farnesylation, but the 50-aa deletion prevents the subsequent processing to mature lamin A. The presence of progerin adversely affects the integrity of the nuclear lamina, resulting in misshapen nuclei and nuclear blebs. We hypothesized that interfering with protein farnesylation would block the targeting of progerin to the nuclear envelope, and we further hypothesized that the mislocalization of progerin away from the nuclear envelope would improve the nuclear blebbing phenotype. To approach this hypothesis, we created a gene-targeted mouse model of HGPS, generated genetically identical primary mouse embryonic fibroblasts, and we then examined the effect of a farnesyltransferase inhibitor on nuclear blebbing. The farnesyltransferase inhibitor mislocalized progerin away from the nuclear envelope to the nucleoplasm, as determined by immunofluoresence microscopy, and resulted in a striking improvement in nuclear blebbing (P < 0.0001 by chi2 statistic). These studies suggest a possible treatment strategy for HGPS.

Novel experimental effects on bone material properties and the pre- and postyield behavior of bones may be independent of bone mineralization.

In this article, we summarize the results of six different tomographic/biomechanical rat studies involving hypophysectomy (Hx), ovariectomy, treatment with rhGH, olpadronate, alendronate, and toxic doses of aluminum and the development of a genetic diabetes in the eSS strain. All these conditions induced some interesting and rarely reported effects on postyield bone strength. These effects were generally related neither to the degree of mineralization or the elastic modulus of the bone tissue nor to the preyield behavior of the bones. In two particular cases (Hx, eSS), the elastic modulus of bone tissue varied independently of its degree of mineralization. These results suggest the involvement of some microstructural factor(s) of bone tissue resistance to crack progression (a postyield feature of bone behavior), rather than to crack initiation (the yield-determining factor) in the corresponding mechanism. Changes in collagen or crystal structure may play that role. These changes are relevant to the mechanism of fracture production during plastic deformation, a feature of bone strength that might be independent from mineralization. Therefore, these changes might help to explain some effects of novel treatments on bone strength unrelated to bone mineralization. This questions the belief that the remaining bone mass in metabolic osteopenias is biologically and mechanically normal.

Absorptiometric assessment of muscle-bone relationships in humans: reference, validation, and application studies.

This report summarizes some preliminary absorptiometric (DXA, QCT/pQCT) studies from our laboratory, supporting the following assumptions. 1. In Homo sapiens at all ages, natural proportionality between DXA-assessed bone mineral mass (bone mineral content, BMC) and muscle mass (lean mass, LM) of the whole body or limbs is specific for ethnicity, gender, and reproductive status, but not for body weight, height, or body mass index. 2. This proportionality is sensitive to many kinds of endocrine-metabolic perturbations. 3. Percentilized or Z-scored charts of the BMC/LM correlations as determined in large samples of healthy individuals can provide a diagnostic reference for evaluating proportionality in different conditions. 4. Employing exclusively DXA, this methodology can be applied to discriminate between "disuse-related" and "metabolic" osteopenias based on the finding of normal or low BMC/LM percentiles or Z-scores respectively, with important therapeutic and monitoring implications.

Micro-CT arthrography: a pilot study for the ex vivo visualization of the rat knee joint.

OBJECTIVE: In our study, we evaluated the potential of micro-CT for the assessment of the rat knee joint using ex vivo micro-CT arthrography. The aims of the study were to introduce the technique of micro-CT arthrography and to visualize the normal anatomy of the rat knee. The secondary aims were the quantification of retropatellar cartilage thickness and the analysis of microstructural cancellous bone parameters within the tibial epiphysis. CONCLUSION: Micro-CT arthrography is a novel technique for the indirect visualization of the distinct features and structural analysis of the rat knee joint. This technique represents an additional imaging and analysis tool in small-animal research.

Heterozygosity for Lmna deficiency eliminates the progeria-like phenotypes in Zmpste24-deficient mice.

Zmpste24 is a metalloproteinase required for the processing of prelamin A to lamin A, a structural component of the nuclear lamina. Zmpste24 deficiency results in the accumulation of prelamin A within cells, a complete loss of mature lamin A, and misshapen nuclear envelopes. Zmpste24-deficient (Zmpste24(-/-)) mice exhibit retarded growth, alopecia, micrognathia, dental abnormalities, osteolytic lesions in bones, and osteoporosis, which are phenotypes shared with Hutchinson-Gilford progeria syndrome, a human disease caused by the synthesis of a mutant prelamin A that cannot undergo processing to lamin A. Zmpste24(-/-) mice also develop muscle weakness. We hypothesized that prelamin A might be toxic and that its accumulation in Zmpste24(-/-) mice is responsible for all of the disease phenotypes. We further hypothesized that Zmpste24(-/-) mice with half-normal levels of prelamin A (Zmpste24(-/-) mice with one Lmna knockout allele) would be subjected to less toxicity and be protected from disease. Thus, we bred and analyzed Zmpste24(-/-)Lmna(+/-) mice. As expected, prelamin A levels in Zmpste24(-/-)Lmna(+/-) cells were significantly reduced. Zmpste24(-/-)Lmna(+/-) mice were entirely normal, lacking all disease phenotypes, and misshapen nuclei were less frequent in Zmpste24(-/-)Lmna(+/-) cells than in Zmpste24(-/-) cells. These data suggest that prelamin A is toxic and that reducing its levels by as little as 50% provides striking protection from disease.

Two new regions of interest to evaluate separately cortical and trabecular BMD in the proximal femur using DXA.

To differentiate changes in trabecular and cortical bone density at a skeletal site bearing body weight, the main goal of this retrospective study was to develop and characterize two new regions of interest (ROIs) for DXA at the hip, one mainly focusing on trabecular bone and another mainly focusing on cortical bone. Specific aims were to maximize the precision of the ROIs and to characterize their usefulness for monitoring age-related bone loss and discriminating controls from fracture cases in a cross-sectional study population and to compare them with earlier ROIs designed by our group. The study used populations from two different previous studies conducted in our laboratory, with one comprising cohorts of healthy premenopausal women, healthy postmenopausal women, and postmenopausal osteoporotic women with at least one spinal fracture (Spine Fx Study) and the other one comprising two cohorts of age-matched postmenopausal women, in whom cases had sustained a hip fracture (Hip Fx study). The new ROI for trabecular bone (CIRCROI) tries to improve on the earlier custom-designed Central ROI, which was also targeted at trabecular bone. CIRCROI consists of an approximate largest circle that can fit inside the femoral proximal metaphysis without touching the superior and inferior endocortical walls. The new ROI for cortical bone (CORTROI) at a site bearing body weight is defined as a horizontal rectangular box crossing the femoral shaft below the lesser trochanter. CORTROI BMD cohort means were significantly higher than all other ROIs, and CIRCROI BMD cohort means were lower than standard ROIs with the exception of Ward's ROI. CIRCROI BMD was highly correlated with total femur BMD ( r=0.94) and Central BMD ( r=0.93), whereas CORTROI BMD correlations were lower (highest with total femur BMD ( r=0.86)). Fracture discrimination odds ratios (ORs) of all ROIs were significant for the Hip Fx Study, with CIRCROI BMD having the highest, and CORTROI BMD the lowest, OR (4.83 and 2.49 per SD, respectively, compared with 3.69 for Ward's ROI as the highest OR of standard ROIs). For the Spine Fx Study, only spinal and trochanteric BMD had significant OR. The new trabecular ROI had good short-term precision, comparable to the standard ROIs at the hip, but improving on that of Ward's triangle, the only standard ROI only including the anterior and posterior cortical walls and therefore more predominantly consisting of trabecular bone than other standard ROIs. The precision of the new cortical ROI was lower than standard DXA ROIs, except for Ward's triangle, but provides unique information on purely cortical bone at a skeletal site bearing body weight.

Zmpste24 deficiency in mice causes spontaneous bone fractures, muscle weakness, and a prelamin A processing defect.

Zmpste24 is an integral membrane metalloproteinase of the endoplasmic reticulum. Biochemical studies of tissues from Zmpste24-deficient mice (Zmpste24(-/-)) have indicated a role for Zmpste24 in the processing of CAAX-type prenylated proteins. Here, we report the pathologic consequences of Zmpste24 deficiency in mice. Zmpste24(-/-) mice gain weight slowly, appear malnourished, and exhibit progressive hair loss. The most striking pathologic phenotype is multiple spontaneous bone fractures-akin to those occurring in mouse models of osteogenesis imperfecta. Cortical and trabecular bone volumes are significantly reduced in Zmpste24(-/-) mice. Zmpste24(-/-) mice also manifested muscle weakness in the lower and upper extremities, resembling mice lacking the farnesylated CAAX protein prelamin A. Prelamin A processing was defective both in fibroblasts lacking Zmpste24 and in fibroblasts lacking the CAAX carboxyl methyltransferase Icmt but was normal in fibroblasts lacking the CAAX endoprotease Rce1. Muscle weakness in Zmpste24(-/-) mice can be reasonably ascribed to defective processing of prelamin A, but the brittle bone phenotype suggests a broader role for Zmpste24 in mammalian biology.

Effect of protein-energy malnutrition in early life on the dimensions and bone quality of the adult rat mandible.

Morphological and biomechanical features of the mandible are negatively affected by protein-energy malnutrition, whose effects are apparently dependent on the time of life of application. The aim here was to investigate, in neonatal rats nursed by dams put on a protein-free diet to depress milk production and thus create a state of protein-energy malnutrition in the offspring, subsequent growth and long-term effects by analyzing mandibular dimensions and bone quality in adulthood. Pregnant Wistar rats were fed a 20% protein diet (control) or a protein-free diet (malnourished) to obtain normal or subnormal milk production, respectively. After weaning, the offspring (males) were fed a 20% protein diet for 70 days. The dimensions of their excised mandibles were measured directly between anatomical points; the geometry and material quality of mandibular bone were assessed by peripheral quantitative computed tomography. Pups suckling from malnourished dams weighed 49.4% of those suckling from control dams at weaning; the actual difference between control and malnourished pups was 25.1g, which persisted until day 91 of age, indicating the absence of catch-up growth. As with body size, the mandibular base length, height and area (an index of mandibular size) were significantly smaller in malnourished than control rats at the end of the study. The mandibular cortical area, volumetric cortical bone mineral content and volumetric cortical bone mineral density assessed by peripheral quantitative computed tomography were similar in both groups of rats at the end of the observation period, but there was a significant reduction in the cortical axial moment of inertia in malnourished rats at this time of postnatal life. These findings suggest that catch-up growth was incomplete in rats malnourished during the suckling period and that the adaptation of mandibular bone architecture to body growth was apparently insufficient to attain normal values, thus not allowing complete compensation in mechanical competence at the end of the study because of an inadequate spatial distribution of resistive material through its cross-section rather than qualitative or quantitative impairment of cortical bone.

Efectos de los bisfosfonatos sobre la eficiencia mecanica de esqueletos normales u osteopenicos / Effects of bisphosphonates on the mechanical competence of normal and osteopenic skeletons

La calidad mecánica de cualquier hueso está determinada por la rigidez (micro-arquitectura, grado de calcificación, microfracturas) y distribución espacial (macro-arquitectura) de su material cortical (en cualquier hueso) y trabecular (sólo en cuerpos vertebrales), resultantes de una combinación de modelación y remodelación óseas. Estos procesos están regulados direccionalmente por el "mecanostato" óseo, sistema retroalimentado cuyo estímulo proviene del uso mecánico regional del esqueleto, y cuyo punto de referencia está permanentemente modulado (no regulado) en forma sistémica por el entorno endocrino-metabólico. Todos los tratamientos medicamentosos en uso para las osteopatías, fragilizantes, incluyendo los bisfosfonatos actúan únicamente modificando no-direccionalmente este último factor. Por eso, sus resultados dependen del grado de estimulación del mecanostato por el uso, y sólo pueden estimarse determinando parßmetros de calidad material y de eficiencia arquitectónica ósea. Los bisfosfonatos mejoran la eficiencia mecánica ósea por inhibir la remodelación (que usualmente arroja balances negativos de masa) afectando, en algún grado (menor para los productos de más cercana generación), la mineralización del material rígido. En nuestro medio contamos con tres productos con efectos biomecánicos óseos positivos demostrados: pamidronato, olpadronato y alendronato. En animales intactos, los tres mejoran la macro-arquitectura ósea a cualquier dosis, aunque el pamidronato deteriora la mineralización del material a dosis muy altas. En animales ooforectomizados o inmovilizados, los tres protegen contra la repercusión biomecánica negativa de la osteopenia impidiendo el deterioro de la calidad del material y sin alterar el diseño arquitectónico óseo. También potencian o prolongan los efectos anabólicos óseos de la hPTH. Sus ventajas terapéuticas se fundan en que ninguno de los tres perturba el accionar del mecanostato, dejando intactos los mecanismos regulatorios de la eficiencia mecánica de la estructura ósea por el uso mecánico, o incluso potenciándolos sistémicamente (efecto anti-catabólico). Esta característica explica que sus efectos dependan mucho del uso mecánico del esqueleto en las regiones que se desea mejorar. Está aun por definirse si una inhibición demasiado intensa o prolongada de la remodelación por los bisfosfonatos pudiera deteriorar o no la reparación de microfracturas que la misma provee, con consecuencias biomecánicas potencialmente...