Dexamethasone downregulates the expression of parathyroid hormone-related protein (PTHrP) in mesenchymal stem cells

Parathyroid hormone-related protein (PTHrP) has been shown to have anabolic effects in women with postmenopausal osteoporosis. PTHrP promotes the recruitment of osteogenic cells and prevents apoptotic death of osteoblasts and osteocytes. The receptor responsible for the effects of PTHrP is the common PTH/PTHrP receptor (PTH1R). Glucocorticoids (GC) are commonly used as drugs to treat inflammatory diseases. Long-term GC treatments are often associated with bone loss which can lead to GC-induced osteoporosis. The aim of this work was to study the effects of the glucocorticoid dexamethasone (Dex) on the expression of PTHrP and PTH1R in adult human mesenchymal stem cells, the progenitor cells of osteoblasts.Adult human mesenchymal stem cells (hMSC) were cultured and differentiated by standard methods. The expression of PTHrP and PTH1R mRNA was assayed by real-time qPCR. The PTHrP release into the culture media was measured by an immunoradiometric assay.Treatment with Dex (10 nM) resulted in an 80% drop in the PTHrP release within 6 h. A 24 h Dex treatment also reduced the expression of PTHrP mRNA by up to 90%. The expression of PTH1R receptor mRNA was simultaneously increased up to 20-fold by 10 nM Dex. The effects of Dex on PTHrP and PTH1R were dose-dependent and experiments with the GC-receptor antagonist mifepristone showed an involvement of GC-receptors in these effects. In addition to the Dex-induced effects on PTHrP and PTH1R, Dex also increased mineralization and the expression of the osteoblast markers Runx2 and alkaline phosphatase. In our studies, we show that dexamethasone decreases the expression of PTHrP and increases the expression of the PTH1R receptor. This could have an impact on PTHrP-mediated anabolic actions on bone and could also affect the responsiveness of circulating PTH. The results indicate that glucocorticoids affect the signalling pathway of PTHrP by regulating both PTHrP and PTH1R expression and these mechanisms could be involved in glucocorticoid-induced osteoporosis.     

Association of the disordered C-terminus of CDC34 with a catalytically bound ubiquitin

Cell division cycle protein 34 (CDC34) is a key E2 ubiquitin (Ub)-conjugating enzyme responsible for the polyubiquitination of proteins controlling the G1/S stages of cell division. The acidic C-terminus of the enzyme is required for this function, although there is little structural information providing details for a mechanism. One logical time point involving the C-terminus is the CDC34-Ub thiolester complex that precedes Ub transfer to a substrate. To examine this, we used a CDC34-Ub disulfide complex that structurally mimics the thiolester intermediate. NMR spectroscopy was used to show that the CDC34 C-terminus is disordered but can intramolecularly interact with the catalytically bound Ub. Using chemical shift perturbation analysis, we mapped two interacting regions on the surface of Ub in the CDC34-Ub complex. The first site comprises a hydrophobic patch (typical of other Ub complexes) that associates with the CDC34 catalytic domain. A novel second site, dependent on the C-terminus of CDC34, comprises a lysine-rich surface (K6, K11, K29, and K33) on the opposite face of Ub. Further, NMR experiments show that this interaction is described by two slowly exchanging states—a compact conformation where the C-terminus of CDC34 interacts with bound Ub and an extended structure where the C-terminus is released. This work provides the first structural details that show how the C-terminus of CDC34 might direct a thiolester-bound Ub to control polyubiquitin chain formation.     

Protein disulfide isomerase isomerizes non-native disulfide bonds in human proinsulin independent of its peptide-binding activity

Protein disulfide isomerase (PDI) supports proinsulin folding as chaperone and isomerase. Here, we focus on how the two PDI functions influence individual steps in the complex folding process of proinsulin. We generated a PDI mutant (PDI-aba'c) where the b' domain was partially deleted, thus abolishing peptide binding but maintaining a PDI-like redox potential. PDI-aba'c catalyzes the folding of human proinsulin by increasing the rate of formation and the final yield of native proinsulin. Importantly, PDI-aba'c isomerizes non-native disulfide bonds in completely oxidized folding intermediates, thereby accelerating the formation of native disulfide bonds. We conclude that peptide binding to PDI is not essential for disulfide isomerization in fully oxidized proinsulin folding intermediates.     

An extract of Artemisia dracunculus L. enhances insulin receptor signaling and modulates gene expression in skeletal muscle in KK-A mice

An ethanolic extract of Artemisia dracunculus L. (PMI 5011) has been observed to decrease glucose and insulin levels in animal models, but the cellular mechanisms by which insulin action is enhanced in vivo are not precisely known. In this study, we evaluated the effects of PMI 5011 to modulate gene expression and cellular signaling through the insulin receptor in skeletal muscle of KK-A^y mice. Eighteen male KK-A^y mice were randomized to a diet (w/w) mixed with PMI 5011 (1%) or diet alone for 8 weeks. Food intake, adiposity, glucose and insulin were assessed over the study, and at study completion, vastus lateralis muscle was obtained to assess insulin signaling parameters and gene expression. Animals randomized to PMI 5011 were shown to have enhanced insulin sensitivity and increased insulin receptor signaling, i.e., IRS-associated PI-3 kinase activity, Akt-1 activity and Akt phosphorylation, in skeletal muscle when compared to control animals (P<.01, P<.01 and P<.001, respectively). Gene expression for insulin signaling proteins, i.e., IRS-1, PI-3 kinase and Glut-4, was not increased, although a relative increase in protein abundance was noted with PMI 5011 treatment. Gene expression for specific ubiquitin proteins and specific 20S proteasome activity, in addition to skeletal muscle phosphatase activity, i.e., PTP1B activity, was significantly decreased in mice randomized to PMI 5011 relative to control. Thus, the data demonstrate that PMI 5011 increases insulin sensitivity and enhances insulin receptor signaling in an animal model of insulin resistance. PMI 5011 may modulate skeletal muscle protein degradation and phosphatase activity as a possible mode of action.     

Cloning, expression and characterization of alcohol dehydrogenases in the silkworm Bombyx mori

Alcohol dehydrogenases (ADH) are a class of enzymes that catalyze the reversible oxidation of alcohols to corresponding aldehydes or ketones, by using either nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP), as coenzymes. In this study, a short-chain ADH gene was identified in Bombyx mori by 5'-RACE PCR. This is the first time the coding region of BmADH has been cloned, expressed, purified and then characterized. The cDNA fragment encoding the BmADH protein was amplified from a pool of silkworm cDNAs by PCR, and then cloned into E. coli expression vector pET-30a(+). The recombinant His-tagged BmADH protein was expressed in E. coli BL21 (DE3), and then purified by metal chelating affinity chromatography. The soluble recombinant BmADH, produced at low-growth temperature, was instrumental in catalyzing the ethanol-dependent reduction of NAD(+), thereby indicating ethanol as one of the substrates of BmADH.     

Rapamycin induces the TGFβ1/Smad signaling cascade in renal mesangial cells upstream of mTOR

The mTOR kinase inhibitor rapamycin (sirolimus) is a drug with potent immunosuppressive and antiproliferative properties. We found that rapamycin induces the TGFβ/Smad signaling cascade in rat mesangial cells (MC) as depicted by the nuclear translocation of phospho-Smads 2, -3 and Smad-4, respectively. Concomitantly, rapamycin increases the nuclear DNA binding of receptor (R)- and co-Smad proteins to a cognate Smad-binding element (SBE) which in turn causes an increase in profibrotic gene expression as exemplified by the connective tissue growth factor (CTGF) and plasminogen activator inhibitor 1 (PAI-1). Using small interfering (si)RNA we demonstrate that Smad 2/3 activation by rapamycin depends on its endogenous receptor FK binding protein 12 (FKBP12). Mechanistically, Smad induction by rapamycin is initiated by an increase in active TGFβ₁ as shown by ELISA and by the inhibitory effects of a neutralizing TGFβ antibody. Using an activin receptor-like kinase (ALK)-5 inhibitor and by siRNA against the TGFβ type II receptor (TGFβ-RII) we furthermore demonstrate a functional involvement of both types of TGFβ receptors. However, rapamycin did not compete with TGFβ for TGFβ-receptor binding as found in radioligand-binding assay. Besides SB203580, a specific inhibitor of the p38 MAPK, the reactive oxygen species (ROS) scavenger N-acetyl-cysteine (NAC) and a cell-permeable superoxide dismutase (SOD) mimetic strongly abrogated the stimulatory effects of rapamycin on Smad 2 and 3 phosphorylation. Furthermore, the rapid increase in dichlorofluorescein (DCF) formation implies that rapamycin mainly acts through ROS. In conclusion, activation of the profibrotic TGFβ/Smad signaling cascade accompanies the immunosuppressive and antiproliferative actions of rapamycin.     

天然来源抗肿瘤化合物作用于MAPK通路的机制

天然来源抗肿瘤活性产物是抗肿瘤药物先导化合物的重要资源。促分裂原活化蛋白激酶(MAPK)通路是细胞内重要的信号转导系统,可以调节细胞的生长、凋亡、黏附、迁移等过程,继而影响肿瘤的发生、发展、侵袭、转移,是潜在的抗肿瘤药物作用靶点之一。本文将对天然来源化合物作用于MAPK信号通路的靶点分子及生物效应进行阐述,为肿瘤的化学预防及治疗提供启发。     

马桑内酯对粘虫体内蛋白质和消化酶的影响

用马桑内酯分别对粘虫采用注射和饲喂处理,48 h后测定粘虫不同部位消化酶活性及蛋白质含量,以探讨马桑有效成分的作用及其杀虫机理.结果显示:(1)注射羟基马桑毒素处理后粘虫组织中蛋白质含量(与丙酮处理比较)均降低,皮组织降低幅度最大为29.47%,饲喂处理后各组织中蛋白质含量均升高,其中皮组织的升高幅度最大为56.87%;但该毒素对粘虫体内蛋白酶和羧酸酯酶的影响不明显.(2)注射与饲喂马桑亭、马桑宁,粘虫体内蛋白质含量均明显比对照处理升高,蛋白酶活性增强,其中饲喂马桑亭处理的粘虫血淋巴中蛋白质含量升高最大为511.49%,蛋白酶活性增强幅度最大为4 640.26%.马桑亭和马桑宁均可使粘虫组织中羧酸酯酶活性降低,其中饲喂马桑宁处理降低幅度最大为82.94%.结果表明:羟基马桑毒素对粘虫体内蛋白质代谢的影响与用药方式有关,马桑亭和马桑宁处理后的粘虫体内蛋白质含量增高、蛋白酶活性增强及酯酶活性降低均达到显著水平.     

The effects of cosolutes on protein dynamics: the reversal of denaturant-induced protein fluctuations by trimethylamine N-oxide

The protein stabilizing effects of the small molecule osmolyte, trimethylamine N-oxide, against chemical denaturant was investigated by NMR spin-relaxation measurements and model-free analysis. In the presence of 0.7 M guanidine hydrochloride increased picosecond-nanosecond dynamics are observed in the protein ribonuclease A. These increased fluctuations occur throughout the protein, but the most significant increases in flexibility occur at positions believed to be the first to unfold. Addition of 0.35 M trimethylamine N-oxide to this destabilized form of ribonuclease results in significant rigidification of the protein backbone as assessed by (1)H-(15)N order parameters. Statistically, these order parameters are the same as those measured in native ribonuclease indicating that TMAO reduces the amplitude of backbone fluctuations in a destabilized protein. These data suggest that TMAO restricts the bond vector motions on the protein energy landscape to resemble those motions that occur in the native protein and points to a relation between stability and dynamics in this enzyme.