Differential effects of endoplasmic reticulum stress-induced autophagy on cell survival

Autophagy is a cellular response to adverse environment and stress, but its significance in cell survival is not always clear. Here we show that autophagy could be induced in the mammalian cells by chemicals, such as A23187, tunicamycin, thapsigargin, and brefeldin A, that cause endoplasmic reticulum stress. Endoplasmic reticulum stress-induced autophagy is important for clearing polyubiquitinated protein aggregates and for reducing cellular vacuolization in HCT116 colon cancer cells and DU145 prostate cancer cells, thus mitigating endoplasmic reticulum stress and protecting against cell death. In contrast, autophagy induced by the same chemicals does not confer protection in a normal human colon cell line and in the non-transformed murine embryonic fibroblasts but rather contributes to cell death. Thus the impact of autophagy on cell survival during endoplasmic reticulum stress is likely contingent on the status of cells, which could be explored for tumor-specific therapy.     

Grb10 and active Raf-1 kinase promote Bad-dependent cell survival

The proapoptotic protein Bad is a key player in cell survival decisions, and is regulated post-translationally by several signaling networks. We expressed Bad in mouse embryonic fibroblasts to sensitize them to apoptosis, and tested cell lines derived from knock-out mice to establish the significance of the interaction between the adaptor protein Grb10 and the Raf-1 protein kinase in anti-apoptotic signaling pathways targeting Bad. When compared with wild-type cells, both Grb10 and Raf-1-deficient cells exhibit greatly enhanced sensitivity to apoptosis in response to Bad expression. Structure-function analysis demonstrates that, in this cellular model, the SH2, proline-rich, and pleckstrin homology domains of Grb10, as well as its Akt phosphorylation site and consequent binding by 14-3-3, are all necessary for its anti-apoptotic functions. As for Raf-1, its kinase activity, its ability to be phosphorylated by Src on Tyr-340/341 and the binding of its Ras-associated domain to the Grb10 SH2 domain are all necessary to promote cell survival. Silencing the expression of either Grb10 or Raf-1 by small interfering RNAs as well as mutagenesis of specific serine residues on Bad, coupled with signaling inhibitor studies, all indicate that Raf-1 and Grb10 are required for the ability of both the phosphatidylinositol 3-kinase/Akt and MAP kinase pathways to modulate the phosphorylation and inactivation of Bad. Because total Raf-1, ERK, and Akt kinase activities are not impaired in the absence of Grb10, we propose that this adapter protein creates a subpopulation of Raf-1 with specific anti-apoptotic activity.     

Repulsive guidance molecule RGMa alters utilization of bone morphogenetic protein (BMP) type II receptors by BMP2 and BMP4

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta superfamily of multifunctional ligands that transduce their signals through type I and II serine/threonine kinase receptors and intracellular Smad proteins. Recently, we identified the glycosylphosphatidylinositol-anchored repulsive guidance molecules RGMa, DRAGON (RGMb), and hemojuvelin (RGMc) as coreceptors for BMP signaling (Babbit, J. L., Huang, F. W., Wrighting, D. W., Xia, Y., Sidis, Y., Samad, T. A., Campagna, J. A., Chung, R., Schneyer, A., Woolf, C. J., Andrews, N. C., and Lin, H. Y. (2006) Nat. Genet. 38, 531-539; Babbit, J. L., Zhang, Y., Samad, T. A., Xia, Y., Tang, J., Schneyer, A., Woolf, C. J., and Lin, H. Y. (2005) J. Biol. Chem. 280, 29820-29827; Samad, T. A., Rebbapragada, A., Bell, E., Zhang, Y., Sidis, Y., Jeong, S. J., Campagna, J. A., Perusini, S., Fabrizio, D. A., Schneyer, A. L., Lin, H. Y., Brivanlou, A. H., Attisano, L., and Woolf, C. J. (2005) J. Biol. Chem. 280, 14122-14129). However, the mechanism by which RGM family members enhance BMP signaling remains unknown. Here, we report that RGMa bound to radiolabeled BMP2 and BMP4 with Kd values of 2.4+/-0.2 and 1.4+/-0.1 nm, respectively. In KGN human ovarian granulosa cells and mouse pulmonary artery smooth muscle cells, BMP2 and BMP4 signaling required BMP receptor type II (BMPRII), but not activin receptor type IIA (ActRIIA) or ActRIIB, based on changes in BMP signaling by small interfering RNA inhibition of receptor expression. In contrast, cells transfected with RGMa utilized both BMPRII and ActRIIA for BMP2 or BMP4 signaling. Furthermore, in BmpRII-null pulmonary artery smooth muscle cells, BMP2 and BMP4 signaling was reduced by inhibition of endogenous RGMa expression, and RGMa-mediated BMP signaling required ActRIIA expression. These findings suggest that RGMa facilitates the use of ActRIIA by endogenous BMP2 and BMP4 ligands that otherwise prefer signaling via BMPRII and that increased utilization of ActRIIA leads to generation of an enhanced BMP signal.     

Nutlin-3 protects kidney cells during cisplatin therapy by suppressing Bax/Bak activation

Nutlins, the newly developed small molecule antagonists of MDM2, activate p53 and induce apoptosis in cancer cells, offering a novel strategy of chemotherapy. Recent studies have further suggested synergistic effects of nutlins with other chemotherapeutic drugs. However, it is unclear whether nutlins increase or decrease the side effects of these drugs in normal non-malignant cells or tissues. Cisplatin is a widely used chemotherapy drug, which has a major side effect of kidney injury. Here we show that Nutlin-3 protected kidney cells against cisplatin-induced apoptosis. The cytoprotective effects of Nutlin-3 were not related to its regulation of p53 or consequent gene expression during cisplatin treatment. Moreover, the protective effects were shown in MDM2-, MDM4-, or p53-deficient cells. On the other hand, Nutlin-3 suppressed mitochondrial events of apoptosis during cisplatin incubation, including Bax activation and cytochrome c release. Nutlin-3 attenuated cisplatin-induced oligomerization of Bax and Bak but not their interactions with Bcl-XL. In isolated mitochondria, Nutlin-3 inhibited cytochrome c release induced by Ca2+, Bim peptide, and recombinant tBid. Importantly, it blocked both Bax and Bak oligomerization under these conditions. Together, the results have uncovered a new pharmacological function of nutlins, i.e. suppression of Bax and Bak, two critical mediators of apoptosis.     

Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non-canonical amino-acid tagging

A major aim of proteomics is the identification of proteins in a given proteome at a given metabolic state. This protocol describes the step-by-step labeling, purification and detection of newly synthesized proteins in mammalian cells using the non-canonical amino acid azidohomoalanine (AHA). In this method, metabolic labeling of newly synthesized proteins with AHA endows them with the unique chemical functionality of the azide group. In the subsequent click chemistry tagging reaction, azide-labeled proteins are covalently coupled to an alkyne-bearing affinity tag. After avidin-based affinity purification and on-resin trypsinization, the resulting peptide mixture is subjected to tandem mass spectrometry for identification. In combination with deuterated leucine-based metabolic colabeling, candidate proteins can be immediately validated. Bioorthogonal non-canonical amino-acid tagging can be combined with any subcellular fractionation, immunopurification or other proteomic method to identify specific subproteomes, thereby reducing sample complexity and enabling the identification of subtle changes in a proteome. This protocol can be completed in 5 days.     

True phosphorus digestibility and the endogenous phosphorus outputs associated with brown rice for weanling pigs measured by the simple linear regression analysis technique

The objectives of this study were to determine true phosphorus (P) digestibility, degradability of phytate-P complex and the endogenous P outputs associated with brown rice feeding in weanling pigs by using the simple linear regression analysis technique. Six barrows with an average initial body weight of 12.5 kg were fitted with a T-cannula and fed six diets according to a 6 × 6 Latin-square design. Six maize starch-based diets, containing six levels of P at 0.80, 1.36, 1.93, 2.49, 3.04, and 3.61 g/kg per kg dry-matter (DM) intake (DMI), were formulated with brown rice. Each experimental period lasted 10 days. After a 7-day adaptation, all faecal samples were collected on days 8 and 9. Ileal digesta samples were collected for a total of 24 h on day 10. The apparent ileal and faecal P digestibility values of brown rice were affected ( P < 0.01) by the P contents in the assay diets. The apparent ileal and faecal P digestibility values increased from − 48.0 to 36.7% and from − 35.6 to 40.0%, respectively, as P content increased from 0.80 to 3.61 g/kg DMI. Linear relationships ( P < 0.05), expressed as g/kg DMI, between the apparent ileal and faecal digestible P and dietary levels of P, suggested that true P digestibility and the endogenous P outputs associated with brown rice feeding could be determined by using the simple regression analysis technique. There were no differences ( P>0.05) in true P digestibility values (57.7 ± 5.4 v. 58.2 ± 5.9%), phytate P degradability (76.4 ± 6.7 v. 79.0 ± 4.4%) and the endogenous P outputs (0.812 ± 0..096 v. 0.725 ± 0.083 g/kg DMI) between the ileal and the faecal levels. The endogenous faecal P output represented 14 and 25% of the National Research Council (1998) recommended daily total and available P requirements in the weanling pig, respectively. About 58% of the total P in brown rice could be digested and absorbed by the weanling pig. Our results suggest that the large intestine of the weanling pigs does not play a significant role in the digestion of P in brown rice. Diet formulation on the basis of total or apparent P digestibility with brown rice may lead to P overfeeding and excessive P excretion in pigs.     

CCN5 Expression in mammals. II. Adult rodent tissues

CCN5 is a secreted heparin- and estrogen-regulated matricellular protein that inhibits vertebrate smooth muscle cell proliferation and motility. CCN5 is expressed throughout murine embryonic development in most organs and tissues. However, after embryonic development is complete, we hypothesized that CCN5 distribution would be largely restricted to small set of tissues, including smooth muscle cells of the arteries, uterus, airway, and digestive tract. Because CCN5 inhibits proliferation of smooth muscle cells in vitro, it might function to prevent excessive growth in vivo. In contrast, another member of the CCN family, CCN2, promotes smooth muscle cell proliferation in vitro, and thus it was expected that its expression levels would be low in uninjured normal adult tissues. Frozen sections from adult tissues and organs were analyzed immunohistochemically using anti-CCN5 and anti-CCN2 antibodies. Both proteins were detected in arteries, the uterus, bronchioles, and the digestive tract as expected, and also in many other tissues including the pancreas, spleen, liver, skeletal muscle, ovary, testis, thymus, brain, olfactory epithelium, and kidney. CCN5 and CCN2 protein was found in smooth muscle, endothelial cells, epithelial cells, skeletal muscle, cells of the nervous system, and numerous other cell types. In many cells, both CCN5 and CCN2 was present in the nucleus. Rather than having opposite patterns of localization, CCN5 and CCN2 often had similar sites of expression. The wide distribution of both CCN5 and CCN2 suggests that both proteins have additional biological functions beyond those previously identified in specific cellular and pathological models.     

Intestinal alkaline phosphatase detoxifies lipopolysaccharide and prevents inflammation in zebrafish in response to the gut microbiota

Vertebrates harbor abundant lipopolysaccharide (LPS) in their gut microbiota. Alkaline phosphatases can dephosphorylate and detoxify the endotoxin component of LPS. Here, we show that expression of the zebrafish intestinal alkaline phosphatase (Iap), localized to the intestinal lumen brush border, is induced during establishment of the gut microbiota. Iap-deficient zebrafish are hypersensitive to LPS toxicity and exhibit the excessive intestinal neutrophil influx characteristic of wild-type zebrafish exposed to LPS. Both of these Iap mutant phenotypes are dependent on Myd88 and Tumor Necrosis Factor Receptor (Tnfr), proteins also involved in LPS sensitivity in mammals. When reared germ-free, the intestines of Iap-deficient zebrafish are devoid of neutrophils. Together, these findings demonstrate that the endogenous microbiota establish the normal homeostatic level of neutrophils in the zebrafish intestine through a process involving Iap, Myd88, and Tnfr. Thus, by preventing inflammatory responses, Iap plays a crucial role in promoting mucosal tolerance to resident gut bacteria.