Shaoli Sun

Effect of low- and high-frequency TENS on Met-enkephalin-Arg-Phe and dynorphin A immunoreactivity in human lumbar CSF

&NA; Transcutaneous nerve stimulation (TENS) treatment was given for 30 min to 37 patients divided into 3 groups of 10 patients and 1 group of 7 patients. Two groups received low‐frequency (2 Hz) and the other 2 groups high‐frequency (100 Hz) stimulation. A diagnostic lumbar cerebrospinal fluid (CSF) sample was obtained immediately before and after stimulation. The CSF samples were subjected to analysis of immunoreactive (ir) opioid peptides, Met‐enkephalin‐Arg‐Phe (MEAP) from preproenkephalin and dynorphin A (Dyn A) from preprodynorphin, respectively. Low frequency TENS applied on the hand and the leg resulted in a marked increase (367%, P < 0.05) of ir‐MEAP but not ir‐Dyn A, whereas high‐frequency (100 Hz) TENS produced a 49% increase in ir‐Dyn A (P < 0.01) but not ir‐MEAP. This is the first report in humans that 2 Hz and 100 Hz peripheral stimulation induces differential release of peptides from preproenkephalin and preprodynorphin, respectively.

Cell surface expression of an endoplasmic reticulum resident heat shock protein gp96 triggers MyD88-dependent systemic autoimmune diseases

Heat shock proteins have been implicated as endogenous activators for dendritic cells (DCs). Without tissue distress or death, these intracellular molecules are inaccessible to surface receptor(s) on DCs, possibly to avoid uncontrolled DC activation and breakdown of immunologic tolerance. We herein addressed this hypothesis in transgenic mice by enforcing cell surface expression of gp96, a ubiquitous heat shock protein of the endoplasmic reticulum. Although a pan-specific promoter is used for transgene expression, neither the expression level nor the tissue distribution of the endogenous gp96 was altered by this maneuver. However, cell surface gp96 induced significant DC activations and spontaneous lupus-like autoimmune diseases, even though the development/functions of lymphocytic compartments were unaltered. Using a bone marrow chimera approach, we further demonstrated that both DC activation and autoimmunity elicited by cell surface gp96 are dependent on the downstream adaptor protein MyD88 for signaling by Toll/IL-1 receptor family. Our study not only established the proinflammatory property of cell surface gp96 in vivo, but also suggested a chronic stimulation of DCs by gp96 as a pathway to initiate spontaneous autoimmune diseases.

Essential roles of grp94 in gut homeostasis via chaperoning canonical Wnt pathway

Increasing evidence points to a role for the protein quality control in the endoplasmic reticulum (ER) in maintaining intestinal homeostasis. However, the specific role for general ER chaperones in this process remains unknown. Herein, we report that a major ER heat shock protein grp94 interacts with MesD, a critical chaperone for the Wnt coreceptor low-density lipoprotein receptor-related protein 6 (LRP6). Without grp94, LRP6 fails to export from the ER to the cell surface, resulting in a profound loss of canonical Wnt signaling. The significance of this finding is demonstrated in vivo in that grp94 loss causes a rapid and profound compromise in intestinal homeostasis with gut-intrinsic defect in the proliferation of intestinal crypts, compromise of nuclear β-catenin translocation, loss of crypt-villus structure, and impaired barrier function. Taken together, our work has uncovered the role of grp94 in chaperoning LRP6-MesD in coordinating intestinal homeostasis, placing canonical Wnt-signaling pathway under the direct regulation of the general protein quality control machinery in the ER.

Inflammasome activation has an important role in the development of spontaneous colitis.

Inflammatory bowel disease (IBD) is characterized for dysregulated intestinal inflammation. Conflicting reports have shown that activation of inflammasome could promote or decrease intestinal inflammation in an acute colitis model, whereas the involvement of inflammasome activation in chronic colitis is poorly understood. In this study, we investigated the role of inflammasome activation in the development of chronic intestinal inflammation by utilizing interleukin-10 (IL-10) knockout (KO) mouse as an animal model, which develops chronic colitis resembling human IBD. We demonstrate the causative link between inflammasome activation and the development of chronic intestinal inflammation. Our results show that mature IL-1β protein levels were significantly increased in all colon sections from IL-10-deficient mice compared with that of wild-type mice. We found that inhibition of inflammasome activities with IL-1 receptor antagonist or caspase-1 inhibitors suppressed IL-1β and IL-17 production from inflamed colon explants. Furthermore, blocking inflammasome activation with caspase-1 inhibitor in vivo significantly ameliorated the spontaneous colitis in IL-10 KO mice. Taken together, these observations demonstrate that inflammasome activation promotes the development of chronic intestinal inflammation.

Gastrointestinal tract epithelial changes associated with taxanes: marker of drug toxicity versus effect.

Background Microscopic findings associated with paclitaxel (Taxol) chemotherapy toxicity were described years ago but whether they are specific for toxicity remains unclear. Further, epithelial changes associated with taxanes can mimic high-grade dysplasia (HGD) in non-neoplastic gastrointestinal (GI) tract mucosa. Similar changes associated with colchicine are only seen in patients with toxicity. Methods GI tract specimens were reviewed (221 total; 93 esophageal, 55 gastric cardiac, 48 oxyntic, 7 antral, 8 small bowel, 6 colonic, 3 appendiceal, 1 anal) from 71 patients (63M, 8F), 38 to 84 years (median, 55 y) undergoing chemotherapy for esophageal, breast, or lung cancer who had all received taxanes at some time [either paclitaxel (Taxol, 55 patients) or docetaxel (Taxotere, 16 patients)]. Epithelial changes (mitotic arrest/ring mitoses, apoptosis) associated with taxanes were graded on a scale of 0 to 3 (0=no mitotic arrest; 1=rare arrest; 2=scattered arrest; 3=striking mitotic arrest and apoptosis). Samples from the patients taken before administration of taxanes were also reviewed; all samples were reviewed without knowledge of the interval between drug doses and the biopsy/resection. Results Five samples had striking mitotic arrest mimicking HGD in non-neoplastic mucosa and were from the esophagus, gastric cardia, gastric body, gastric antrum, and appendix of 5 separate patients. All 5 had GI tract samples obtained 1 to 3 days after taxane administration. These patients had all taken Taxol (rather than Taxotere). On follow-up, in 3/5 patients with samples 1 day posttreatment, 1 had acute appendicitis (died 180 d postappendectomy), 1 died a day later of metastases, and 1 was asymptomatic (alive with metastatic disease at 126 d postbiopsy). The remaining 2 died of metastases at 90 and 210 days postbiopsy with no signs of drug toxicity at any time. Conclusions In contrast to colchicine-associated changes in non-neoplastic mucosa, the mitotic arrest mimicking HGD seen in GI tract specimens after taxane administration is not specific for toxicity, but may also reflect taxane effect. It can be encountered in asymptomatic patients who have recently had medication. If these findings are seen histologically, they merit correlation with the clinical impression, and should not be interpreted as toxicity in isolation.

TLR4 Hyperresponsiveness via Cell Surface Expression of Heat Shock Protein gp96 Potentiates Suppressive Function of Regulatory T Cells

As one of the main mediators of the endoplasmic reticulum unfolded protein response, heat shock protein gp96 is also an obligate chaperone for multiple TLRs including TLR4. We demonstrated recently that enforced cell surface expression of gp96 in a transgenic (Tg) mouse (96tm-Tg) conferred hyperresponsiveness to LPS and induced TLR4-dependent lupus-like autoimmune diseases. In this study, we investigated the function of CD4+CD25+ Foxp3+ regulatory T cells (Treg) in these mice in light of the important roles of Treg in the maintenance of peripheral tolerance against self-Ag as well as the increasing appreciation of TLR signaling on the regulation of Treg. We found that the development of Treg was not impaired in 96tm-Tg mice. Contrary to the prediction of dampened Treg activity, we discovered that the suppressive functions of Treg were increased in 96tm-Tg mice. Inactivation of Treg during the neonatal stage of life exacerbated not only organ-specific diseases but also systemic autoimmune diseases. By crossing 96tm-Tg mice into the TLR4 null background, we demonstrated the critical roles of TLR4 in the amplification of Treg suppressive function. These findings illustrate that gp96 plays dual roles in regulating immune responses by augmenting proinflammatory responses and inducing Treg function, both of which are dependent on its ability to chaperone TLR4. Our study provides strong support to the notion of compensatory Treg activation by TLR ligation to dampen inflammation and autoimmune diseases.

Immune chaperone gp96 drives the contributions of macrophages to inflammatory colon tumorigenesis

Macrophages are important drivers in the development of inflammation-associated colon cancers, but the mechanistic underpinnings for their contributions are not fully understood. Furthermore, Toll-like receptors have been implicated in colon cancer, but their relevant cellular sites of action are obscure. In this study, we show that the endoplasmic reticulum chaperone gp96 is essential in tumor-associated macrophages (TAM) to license their contributions to inflammatory colon tumorigenesis. Mice where gp96 was genetically deleted in a macrophage-specific manner exhibited reduced colitis and inflammation-associated colon tumorigenesis. Attenuation of colon cancer in these mice correlated strikingly with reduced mutation rates of β-catenin, increased efficiency of the DNA repair machinery, and reduced expression of proinflammatory cytokines, including interleukin (IL)-17 and IL-23 in the tumor microenvironment. The genotoxic nature of TAM-associated inflammation was evident by increased expression of genes in the DNA repair pathway. Our work deepens understanding of how TAM promote oncogenesis by altering the molecular oncogenic program within epithelial cells, and it identifies gp96 as a lynchpin chaperone needed in TAM to license their function and impact on expression of critical inflammatory cytokines in colon tumorigenesis.

Molecular Profiling of Multiple Human Cancers Defines an Inflammatory Cancer-Associated Molecular Pattern and Uncovers KPNA2 as a Uniform Poor Prognostic Cancer Marker

Background Immune evasion is one of the recognized hallmarks of cancer. Inflammatory responses to cancer can also contribute directly to oncogenesis. Since the immune system is hardwired to protect the host, there is a possibility that cancers, regardless of their histological origins, endow themselves with a common and shared inflammatory cancer-associated molecular pattern (iCAMP) to promote oncoinflammation. However, the definition of iCAMP has not been conceptually and experimentally investigated. Methods and Findings Genome-wide cDNA expression data was analyzed for 221 normal and 324 cancer specimens from 7 cancer types: breast, prostate, lung, colon, gastric, oral and pancreatic. A total of 96 inflammatory genes with consistent dysregulation were identified, including 44 up-regulated and 52 down-regulated genes. Protein expression was confirmed by immunohistochemistry for some of these genes. The iCAMP contains proteins whose roles in cancer have been implicated and others which are yet to be appreciated. The clinical significance of many iCAMP genes was confirmed in multiple independent cohorts of colon and ovarian cancer patients. In both cases, better prognosis correlated strongly with high CXCL13 and low level of GREM1, LOX, TNFAIP6, CD36, and EDNRA. An “Inflammatory Gene Integrated Score” was further developed from the combination of 18 iCAMP genes in ovarian cancer, which predicted overall survival. Noticeably, as a selective nuclear import protein whose immuno-regulatory function just begins to emerge, karyopherin alpha 2 (KPNA2) is uniformly up-regulated across cancer types. For the first time, the cancer-specific up-regulation of KPNA2 and its clinical significance were verified by tissue microarray analysis in colon and head-neck cancers. Conclusion This work defines an inflammatory signature shared by seven epithelial cancer types and KPNA2 as a consistently up-regulated protein in cancer. Identification of iCAMP may not only serve as a novel biomarker for prognostication and individualized treatment of cancer, but also have significant biological implications.

GP96 is a GARP chaperone and controls regulatory T cell functions.

Molecular chaperones control a multitude of cellular functions via folding chaperone-specific client proteins. CD4+FOXP3+ Tregs play key roles in maintaining peripheral tolerance, which is subject to regulation by multiple molecular switches, including mTOR and hypoxia-inducible factor. It is not clear whether GP96 (also known as GRP94), which is a master TLR and integrin chaperone, controls Treg function. Using murine genetic models, we demonstrated that GP96 is required for Treg maintenance and function, as loss of GP96 resulted in instability of the Treg lineage and impairment of suppressive functions in vivo. In the absence of GP96, Tregs were unable to maintain FOXP3 expression levels, resulting in systemic accumulation of pathogenic IFN-γ-producing and IL-17-producing T cells. We determined that GP96 serves as an essential chaperone for the cell-surface protein glycoprotein A repetitions predominant (GARP), which is a docking receptor for latent membrane-associated TGF-β (mLTGF-β). The loss of both GARP and integrins on GP96-deficient Tregs prevented expression of mLTGF-β and resulted in inefficient production of active TGF-β. Our work demonstrates that GP96 regulates multiple facets of Treg biology, thereby placing Treg stability and immunosuppressive functions strategically under the control of a major stress chaperone.

Endoplasmic reticulum heat shock protein gp96 maintains liver homeostasis and promotes hepatocellular carcinogenesis.

BACKGROUND & AIMS gp96, or grp94, is an endoplasmic reticulum (ER)-localized heat shock protein 90 paralog that acts as a protein chaperone and plays an important role for example in ER homeostasis, ER stress, Wnt and integrin signaling, and calcium homeostasis, which are vital processes in oncogenesis. However, the cancer-intrinsic function of gp96 remains controversial. METHODS We studied the roles of gp96 in liver biology in mice via an Albumin promoter-driven Cre recombinase-mediated disruption of gp96 gene, hsp90b1. The impact of gp96 status on hepatic carcinogenesis in response to diethyl-nitrosoamine (DENA) was probed. The roles of gp96 on human hepatocellular carcinoma cells (HCC) were also examined pharmacologically with a targeted gp96 inhibitor. RESULTS We demonstrated that gp96 maintains liver development and hepatocyte function in vivo, and its loss genetically promotes adaptive accumulation of long chain ceramides, accompanied by steatotic regeneration of residual gp96+ hepatocytes. The need for compensatory expansion of gp96+ cells in the gp96- background predisposes mice to develop carcinogen-induced hepatic hyperplasia and cancer from gp96+ but not gp96- hepatocytes. We also found that genetic and pharmacological inhibition of gp96 in human HCCs perturbed multiple growth signals, and attenuated proliferation and expansion. CONCLUSIONS gp96 is a pro-oncogenic chaperone and an attractive therapeutic target for HCC.