RESUMEN
Inorganic phosphate (Pi) is a critical determinant of calcification, and its concentration is regulated by alkaline phosphatase (ALP) and Pit1. ALP is a key regulator of osteogenic calcification and acts by modulating local inorganic phosphate (Pi) concentrations through hydrolyzing pyrophosphate in the extracellular matrix (ECM). Pit1, a sodium-dependent phosphate transporter, regulates calcification via facilitating phosphate uptake within the cells. To investigate whether zinc differentially regulates osteoblastic and vascular calcifications, we examined ALP activity and Pit1 in osteoblastic and vascular smooth muscle cells (VSMCs). Our findings demonstrate that calcification in osteoblastic MC3T3-E1 cells is decreased via diminished ALP action under zinc deficiency. In contrast, zinc-deficiency-induced calcification in VSMCs is independent of ALP action, as demonstrated by very weak ALP activity and expression in calcified VSMCs. In zinc-deficient A7r5 VSMC, P accumulation increased with increasing Na phosphate concentration (3-7 mM) but not with ß-GP treatment, which requires ALP activity to generate Pi. Ca deposition also increased with Na phosphate in a dose-dependent manner; in contrast, ß-GP did not affect Ca deposition. In osteoblastic cells, Pit1 expression was not affected by zinc treatments. In contrast, Pit1 expression is highly upregulated in A7r5 VSMC under zinc deficiency. Using phosphonoformic acid, a competitive inhibitor of Pit1, we showed that calcification is inhibited in both A7r5 and MC3T3-E1 cells, indicating a requirement for Pit1 in both calcifications. Moreover, the downregulation of VSMC markers under zinc deficiency was restored by blocking Pit1. Taken together, our results imply that zinc-deficiency-induced calcification in VSMC is independent of ALP action in contrast to osteoblastic calcification. Moreover, Pit1 expression in VSMCs is a target for zinc deficiency and may mediate the inhibition of VSMC marker expression under zinc deficiency.
Asunto(s)
Desnutrición , Calcificación Vascular , Humanos , Regulación hacia Arriba , Músculo Liso Vascular , Fosfatasa Alcalina , Zinc/farmacologíaRESUMEN
BACKGROUND: The accelerated proliferation of vascular smooth muscle cells (VSMCs) is a contributor for atherosclerosis by thickening the vascular wall. Since zinc modulation of VSMC proliferation has not been clarified, this study investigated whether zinc affects VSMC proliferation. METHODS AND RESULTS: Both a rat aorta origin vascular smooth muscle cell line (A7r5 VSMCs) and primary VSMCs which were collected from rat aorta (pVSMCs) were cultured with zinc (0-50 µM Zn) for short- (≤12 d) and long-term (28 d) periods under normal non-calcifying (0 or 1 mM P) or calcifying (>2 mM P) P conditions. Mouse vascular endothelial cells (MS I cells) were also cultured (under 0-50 µM Zn and 10 mM P for 20 d) to compare with VSMC cultures. While during short-term culture of VSMCs, zinc deprivation decreased cell proliferation in a zinc-concentration manner both under non-calcifying and calcifying conditions in A7r5 and pVSMCs (P < 0.05), during long-term cultures (28 d), A7r5 VSMC proliferation was inversely related to medium zinc concentration under normal physiological P conditions (regression coefficient r(2) = -0.563, P = 0.012). The anti-cell proliferative effect of zinc supplementation (>50 µM) was VSMC-specific. Long-term (35 d), low zinc treatment down-regulated JNK expression and activation, while not affecting ERK1/2 MAPK signaling in A7r5 VSMCs. CONCLUSION: The results showed that chronic zinc deprivation accelerated VSMC proliferation, perhaps due to down-regulation of MAPK-JNK signaling, and that the anti-cell proliferative role of zinc is VSMC-specific. The findings suggested that zinc may have anti-VSMC proliferative properties in atherosclerosis.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Proteína Quinasa 9 Activada por Mitógenos/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Zinc/deficiencia , Zinc/farmacología , Animales , Aorta/citología , Calcio/metabolismo , Medios de Cultivo/farmacología , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Cultivo Primario de Células , Ratas , Factor de Transcripción STAT3/metabolismo , Factores de TiempoRESUMEN
SCOPE: Zinc is implicated as an activator for bone formation, however, its influence on bone calcification has not been reported. This study examined how zinc regulates the bone matrix calcification in osteoblasts. METHODS AND RESULTS: Two osteoblastic MC3T3-E1 cell subclones (SC 4 and SC 24 as high and low osteogenic differentiation, respectively) were cultured in normal osteogenic (OSM), Zinc deficient (Zn-, 1 µM), or adequate (Zn+, 15 µM) media up to 20 days. Cells (SC 4) were also supplemented with (50 µg/mL) or no ascorbic acid (AA) in combination with Zinc treatment. Zn- decreased collagen synthesis and matrix accumulation. Although AA is essential for collagen formation, its supplementation could not compensate for Zinc deficiency-induced detrimental effects on extracellular matrix mineralization. Zn- also decreased the medium and cell layer alkaline phosphatase ALP activity. This decreased ALP activity might cause the decrease of Pi accumulation in response to Zn-, as measured by von Kossa staining. Ca deposition in cell layers, measured by Alizarin red S staining, was also decreased by Zn(-) . CONCLUSION: Our findings suggest that zinc deprivation inhibits extracellular matrix calcification in osteoblasts by decreasing the synthesis and activity of matrix proteins, type I collagen and ALP, and decreasing Ca and Pi accumulation. Therefore zinc deficiency can be considered as risk factor for poor extracellular matrix calcification.
Asunto(s)
Calcificación Fisiológica/efectos de los fármacos , Matriz Extracelular/metabolismo , Osteoblastos/efectos de los fármacos , Zinc/deficiencia , Fosfatasa Alcalina/metabolismo , Animales , Ácido Ascórbico/farmacología , Calcio/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Colágeno Tipo I/metabolismo , Matriz Extracelular/efectos de los fármacos , Ratones , Zinc/farmacología , Zinc/fisiologíaRESUMEN
Diosgenin, a steroid saponin extracted from the root of wild yam (Dioscorea villossa) is claimed to have osteogenic property. However, detailed studies providing evidence to this claim have not been fully undertaken. In this study, we investigated the effect of diosgenin on the osteogenesis of murine MC3T3-E1 osteoblastic cells. Cells were cultured with varying levels of diosgenin (0-10 µM) within 25 days of bone formation period. Diosgenin was found to stimulate proliferation within the range of 0.01-5 µM using MTT assay. The medium and cellular levels of Type 1 collagen and alkaline phosphatase (ALP), both of which are major bone matrix proteins, increased within the low range of diosgenin concentration (>0-3 µM), and this pattern was further confirmed by collagen and ALP staining of the extracellular matrix (ECM). The cellular protein expression of ALP and collagen Type 1 was also increased at 0.1-1 µM diosgenin treatment as analyzed by Western blot. Calcium deposition within the ECM also showed the same pattern as assessed by Alizarin Red S and Von Kossa staining. Bone-specific transcription factor runt-related transcription factor 2 (Runx2) and Runx2-regulated osteopontin protein expressions were induced at low concentration (0.1-1 µM) and again decreased with high diosgenin concentrations. Based on our findings, our study suggests that diosgenin can enhance bone formation by stimulating the synthesis and secretion of Type 1 collagen and ALP and bone marker proteins Runx2 and osteopontin expression. The increased levels of these marker proteins, in turn, can increase the formation of calcium deposits within the ECM thereby increasing bone formation.
Asunto(s)
Matriz Ósea/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Diosgenina/farmacología , Osteoblastos/metabolismo , Osteogénesis/efectos de los fármacos , Fosfatasa Alcalina/biosíntesis , Animales , Calcificación Fisiológica/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Colágeno Tipo I/biosíntesis , Matriz Extracelular/metabolismo , Ratones , Osteoblastos/efectos de los fármacos , Osteopontina/biosíntesisRESUMEN
The lysozymes encoded by bacteriophage T7 and K11 are both bifunctional enzymes sharing an extensive sequence homology (75%). The constructions of chimeric lysozymes were carried out by swapping the N-terminal and C-terminal domains between phage T7 and K11 lysozymes. This technique generated two chimeras, T7K11-lysozyme (N-terminal T7 domain and C-terminal K11 domain) and K11T7-lysozyme (N-terminal K11 domain and C-terminal T7 domain), which are both enzymatically active. The amidase activity of T7K11-lysozyme is comparable with the parental enzymes while K11T7-lysozyme exhibits an activity that is approximately 45% greater than the wild-type lysozymes. Moreover, these chimeric constructs have optimum pH of 7.2-7.4 similar to the parental lysozymes but exhibit greater thermal stabilities. On the other hand, the chimeras inhibit transcription comparable with the parental lysozymes depending on the source of their N-terminals. Taken together, our results indicated that domain swapping technique localizes the N-terminal region as the domain responsible for the transcription inhibition specificity of the wild type T7 and K11 lysozymes. Furthermore, we were able to develop a simple and rapid purification scheme in purifying both the wild-type and chimeric lysozymes.