RESUMEN
Ca2+ oscillation is a system-level property of the cellular Ca2+-handling machinery and encodes diverse physiological and pathological signals. The present study tests the hypothesis that Ca2+ oscillations play a vital role in maintaining the stemness of liver cancer stem cells (CSCs), which are postulated to be responsible for cancer initiation and progression. We found that niche factor-stimulated Ca2+ oscillation is a signature feature of CSC-enriched Hep-12 cells and purified α2δ1+ CSC fractions from hepatocellular carcinoma cell lines. In Hep-12 cells, the Ca2+ oscillation frequency positively correlated with the self-renewal potential. Using a newly developed high signal, endoplasmic reticulum (ER) localized Ca2+ sensor GCaMP-ER2, we demonstrated CSC-distinctive oscillatory ER Ca2+ release controlled by the type 2 inositol 1,4,5-trisphosphate receptor (IP3R2). Knockdown of IP3R2 severely suppressed the self-renewal capacity of liver CSCs. We propose that targeting the IP3R2-mediated Ca2+ oscillation in CSCs might afford a novel, physiologically inspired anti-tumor strategy for liver cancer.
Asunto(s)
Calcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Células Madre Neoplásicas/metabolismo , Adenosina Trifosfato/farmacología , Animales , Línea Celular Tumoral , Autorrenovación de las Células , Retículo Endoplásmico/química , Retículo Endoplásmico/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Humanos , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inhibidores , Receptores de Inositol 1,4,5-Trifosfato/genética , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/uso terapéutico , Trasplante HeterólogoRESUMEN
Transmembrane and coiled-coil domains 1 (TMCO1) is a recently identified Ca2+ leak channel in the endoplasmic reticulum. TMCO1 dysfunction in humans is associated with dysmorphism, mental retardation, glaucoma and the occurrence of cancer. Here we show an essential role of TMCO1 in osteogenesis mediated by local Ca2+/CaMKII signaling in osteoblasts. TMCO1 levels were significantly decreased in bone from both osteoporosis patients and bone-loss mouse models. Tmco1-/- mice exhibited loss of bone mass and altered microarchitecture characteristic of osteoporosis. In the absence of TMCO1, decreased HDAC4 phosphorylation resulted in nuclear enrichment of HADC4, which leads to deacetylation and degradation of RUNX2, the master regulator of osteogenesis. We further demonstrate that TMCO1-mediated Ca2+ leak provides local Ca2+ signals to activate the CaMKII-HDAC4-RUNX2 signaling axis. The establishment of TMCO1 as a pivotal player in osteogenesis uncovers a novel potential therapeutic target for ameliorating osteoporosis.
Asunto(s)
Canales de Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteínas de la Membrana/metabolismo , Osteoblastos/metabolismo , Osteoporosis/fisiopatología , Anciano , Animales , Canales de Calcio/genética , Señalización del Calcio , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Femenino , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Mutantes , Persona de Mediana Edad , Osteogénesis/fisiología , Osteoporosis/metabolismo , Ovariectomía , Estabilidad Proteica , Transducción de SeñalRESUMEN
Comamonas strain CNB-1 was isolated from a biological reactor treating wastewater from a p-chloronitrobenzene production factory. Strain CNB-1 used p-chloronitrobenzene as sole source of carbon, nitrogen, and energy. A 2-aminophenol 1,6-dioxygenase was purified from cells of strain CNB-1. The purified 2-aminophenol 1,6-dioxygenase had a native molecular mass of 130 kDa and was composed of alpha- and beta-subunits of 33 and 38 kDa, respectively. This enzyme is different from currently known 2-aminophenol 1,6-dioxygenases in that it: (a) has a higher affinity for 2-amino-5-chlorophenol (K(m)=0.77 microM) than for 2-aminophenol (K(m)=0.89 microM) and (b) utilized protocatechuate as a substrate. These results suggested that 2-amino-5-chlorophenol, an intermediate during p-chloronitrobenzene degradation, is the natural substrate for this enzyme. N-terminal amino acids of the alpha- and beta-subunits were determined to be T-V-V-S-A-F-L-V and M-Q-G-E-I-I-A-E, respectively. A cosmid library was constructed from the total DNA of strain CNB-1 and three clones (BG-1, BG-2, and CG-13) with 2-aminophenol 1,6-dioxygenase activities were obtained. DNA sequencing of clone BG-2 revealed a 15-kb fragment that contained two ORFs, ORF9 and ORF10, with N-terminal amino acid sequences identical to those of the beta- and alpha-subunits, respectively, from the purified 2-aminophenol 1,6-dioxygenase. The enzyme was actively synthesized when the genes coding for the ORF9 and ORF10 were cloned into Escherichia coli.
Asunto(s)
Comamonas/enzimología , Comamonas/metabolismo , Dioxigenasas/genética , Dioxigenasas/metabolismo , Nitrobencenos/metabolismo , Secuencia de Aminoácidos , Aminofenoles/metabolismo , Biodegradación Ambiental , Clorofenoles/metabolismo , Clonación Molecular , Comamonas/genética , Comamonas/aislamiento & purificación , Cósmidos , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Dioxigenasas/química , Dioxigenasas/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/metabolismo , Biblioteca de Genes , Genes Bacterianos , Hidroxibenzoatos/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Sistemas de Lectura Abierta , Subunidades de Proteína/química , Subunidades de Proteína/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Análisis de Secuencia de ADN , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Microbiología del AguaRESUMEN
The sulfur oxygenase/reductase (SOR) of Acidianus strain S5 was purified and characterized after expressing the SOR gene in a recombinant strain of Escherichia coli. The N-terminal sequence of the purified SOR protein was the same as the deduced amino acid sequence from previously cloned SOR genes. Enzymatic studies indicated that the SOR catalyzed the conversion of elemental sulfur (S(o)) to sulfite, thiosulfate, and sulfide. The optimal pH and temperature were 5.0 and 70 degrees C, respectively. Comparison of this SOR and that of A. ambivalens revealed several differences between these two SORs. The most striking difference is that the SOR of Acidianus S5 had maximal activity at acidic pH. By application of anti-SOR serum and the Western blot technique, it was found that SOR proteins existed in A. brierleyi and in Acidianus S5 cells cultivated with thiosulfate as the sole energy source, indicating that SOR may also play a role in thiosulfate metabolism.