RESUMO
Osteopontin (OPN) and Bone Sialoprotein (BSP), abundantly expressed by osteoblasts and osteoclasts, appear to have important, partly overlapping functions in bone. In gene-knockout (KO, -/-) models of either protein and their double (D)KO in the same CD1/129sv genetic background, we analyzed the morphology, matrix characteristics, and biomechanical properties of femur bone in 2 and 4 month old, male and female mice. OPN-/- mice display inconsistent, perhaps localized hypermineralization, while the BSP-/- are hypomineralized throughout ages and sexes, and the low mineralization of young DKO mice recovers with age. The higher contribution of primary bone remnants in OPN-/- shafts suggests a slow turnover, while their lower percentage in BSP-/- indicates rapid remodeling, despite FTIR-based evidence in this genotype of a high maturity of the mineralized matrix. In 3-point bending assays, OPN-/- bones consistently display higher Maximal Load, Work to Max. Load and in young mice Ultimate Stress, an intrinsic characteristic of the matrix. Young male and old female BSP-/- also display high Work to Max. Load along with low Ultimate Stress. Principal Component Analysis confirms the major role of morphological traits in mechanical competence, and evidences a grouping of the WT phenotype with the OPN-/- and of BSP-/- with DKO, driven by both structural and matrix parameters, suggesting that the presence or absence of BSP has the most profound effects on skeletal properties. Single or double gene KO of OPN and BSP thus have multiple distinct effects on skeletal phenotypes, confirming their importance in bone biology and their interplay in its regulation.
Assuntos
Sialoproteína de Ligação à Integrina , Camundongos Knockout , Osteopontina , Animais , Osteopontina/genética , Osteopontina/metabolismo , Feminino , Masculino , Camundongos , Sialoproteína de Ligação à Integrina/genética , Sialoproteína de Ligação à Integrina/metabolismo , Fenômenos Biomecânicos , Osso e Ossos/metabolismo , Densidade Óssea/fisiologia , Densidade Óssea/genética , Fêmur/metabolismo , Calcificação Fisiológica/fisiologia , Calcificação Fisiológica/genéticaRESUMO
Medial artery calcification, a hallmark of type 2 diabetes mellitus and chronic kidney disease (CKD), is known as an independent risk factor for cardiovascular mortality and morbidity. Hyperphosphatemia associated with CKD is a strong stimulator of vascular calcification but the molecular mechanisms regulating this process remain not fully understood. We showed that calcification was induced after exposing Sprague-Dawley rat aortic explants to high inorganic phosphate level (Pi , 6 mM) as examined by Alizarin red and Von Kossa staining. This calcification was associated with high Tissue-Nonspecific Alkaline Phosphatase (TNAP) activity, vascular smooth muscle cells de-differentiation, manifested by downregulation of smooth muscle 22 alpha (SM22α) protein expression which was assessed by immunoblot analysis, immunofluorescence, and trans-differentiation into osteo-chondrocyte-like cells revealed by upregulation of Runt related transcription factor 2 (Runx2), TNAP, osteocalcin, and osteopontin mRNA levels which were determined by quantitative real-time PCR. To unravel the possible mechanism(s) involved in this process, microRNA (miR) expression profile, which was assessed using TLDA technique and thereafter confirmed by individual qRT-PCR, revealed differential expression 10 miRs, five at day 3 and 5 at day 6 post Pi treatment versus control untreated aortas. At day 3, miR-200c, -155, 322 were upregulated and miR-708 and 331 were downregulated. After 6 days of treatment, miR-328, -546, -301a were upregulated while miR-409 and miR-542 were downregulated. Our results indicate that high Pi levels trigger aortic calcification and modulation of certain miRs. These observations suggest that mechanisms regulating aortic calcification might involve miRs, which warrant further investigations in future studies.
Assuntos
Calcificação Fisiológica/genética , Hiperfosfatemia/genética , MicroRNAs/genética , Insuficiência Renal Crônica/genética , Fosfatase Alcalina/genética , Animais , Desdiferenciação Celular/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hiperfosfatemia/fisiopatologia , Proteínas dos Microfilamentos/genética , Proteínas Musculares/genética , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Osteocalcina/genética , Fosfatos/farmacologia , Ratos , Insuficiência Renal Crônica/fisiopatologiaRESUMO
Fetal bovine mandible-derived osteoblasts were cultured for the purpose of obtaining a spatiotemporal assessment of bone matrix protein expression during in vitro differentiation. The results obtained from electron microscopic, immunohistological, biochemical, and molecular biological analyses indicated that these primary cultured osteoblasts produce an abundant extracellular matrix which mineralizes during a 14-day culture period. During this process, a restricted, spatiotemporal pattern of bone sialoprotein expression was indicated by immunohistological and molecular evaluations. To test the possibility that bone sialoprotein promoted the continued morphodifferentiation of osteoblastic cells, cultures were grown in the presence of anti-bone sialoprotein antibodies known to interfere with cell-bone sialoprotein attachment. Compared with cultures grown in the presence of normal rabbit serum (1:150), cultures grown in the media containing anti-bone sialoprotein antibody (1:150) failed to mineralize as demonstrated by von Kossa staining and failed to express osteocalcin and osteopontin as shown by the reverse transcription polymerase chain reaction. These results contribute to the growing evidence that bone sialoprotein is an important determinant of osteoblast differentiation and bone formation. Matrix protein-cell interactions may be examined using this spatiotemporally defined model.