Samuel Shao-Min Zhang

STAT3 deletion during hematopoiesis causes Crohn's disease-like pathogenesis and lethality : a critical role of STAT3 in innate immunity

Signal transducer and activator of transcription 3 (STAT3) is a key transcriptional mediator for many cytokines and is essential for normal embryonic development. We have generated a unique strain of mice with tissue-specific disruption of STAT3 in bone marrow cells during hematopoiesis. This specific STAT3 deletion causes death of these mice within 4–6 weeks after birth with Crohns disease-like pathogenesis in both the small and large intestine, including segmental inflammatory cell infiltration, ulceration, bowel wall thickening, and granuloma formation. Deletion of STAT3 causes significantly increased cell autonomous proliferation of cells of the myeloid lineage, both in vivo and in vitro. Most importantly, Stat3 deletion during hematopoiesis causes overly pseudoactivated innate immune responses. Although inflammatory cytokines, including tumor necrosis factor α and IFN-γ, are overly produced in these mice, the NAPDH oxidase activity, which is involved in antimicrobial and innate immune responses, is inhibited. The signaling responses to lipopolysaccharide are changed in the absence of STAT3, leading to enhanced NF-κB activation. Our results suggest a model in which STAT3 has critical roles in the development and regulation of innate immunity, and deletion of STAT3 during hematopoiesis results in abnormalities in myeloid cells and causes Crohns disease-like pathogenesis.

Cardiomyocyte-restricted knockout of STAT3 results in higher sensitivity to inflammation, cardiac fibrosis, and heart failure with advanced age

Cytokines and inflammation have been implicated in the pathogenesis of heart failure. For example, IL-6 family cytokines and the gp130 receptor play important roles in cardiac myocyte survival and hypertrophy. Signal transducer and activator of transcription 3 (STAT3) is a major signaling protein that is activated through gp130. We have created mice with a cardiomyocyte-restricted deletion of STAT3. As measured by serial echocardiograms, mice with cardiac specific deletion of STAT3 are significantly more susceptible to cardiac injury after doxorubicin treatment than age-matched controls. Intriguingly, STAT3 appears to have a critical role in protection of inflammation-induced heart damage. STAT3-deficient mice treated with lipopolysaccharide demonstrated significantly more apoptosis than their WT counterparts. At the cellular level, cardiomyocytes with STAT3 deleted secrete significantly more tumor necrosis factor α in response to lipopolysaccharide than those with WT STAT3. Furthermore, histologic examination of the cardiomyocyte-restricted STAT3-deficient mice reveals a dramatic increase in cardiac fibrosis in aged mice. Although no overt signs of heart failure are present in young STAT3-deficient mice, they spontaneously develop heart dysfunction with advancing age. These results indicate the crucial functions of STAT3 in cardiomyocyte resistance to inflammation and other acute injury and in pathogenesis of age-related heart failure.

STAT3-Mediated Signaling Dysregulates Lung Fibroblast-Myofibroblast Activation and Differentiation in UIP/IPF

STAT3 is a latent transcription factor that plays a role in regulating fibroblast function in fibrotic lung diseases. To further understand the role of STAT3 in the phenotypic divergence and function of human lung fibroblasts (LFs), we investigated the effect of basal and cytokine-induced STAT3 activity on indices of LF differentiation and activation, including expression of α-smooth muscle actin (α-SMA), collagen, and adhesion molecules Thy-1/CD90 and α(v) β(3) and β(5) integrins. We identified a population of fibroblasts from usual interstitial pneumonia (UIP)/idiopathic pulmonary fibrosis (IPF) lungs characterized by constitutively phosphorylated STAT3, lower proliferation rates, and diminished expression of α-SMA, Thy-1/CD90, and β(3) integrins compared with control LFs. Staining of UIP lung biopsy specimens demonstrated that phosphorylated STAT3 was not present in α-SMA-positive fibroblastic foci but was observed in the nuclei of cells located in the areas of dense fibrosis. STAT3 activation in LFs did not significantly influence basal or transforming growth factor β(1)-induced collagen I expression but inhibited expression of α-SMA, Thy-1/CD90, and αv β(3) integrins. Suppression of STAT3 signaling diminished resistance of IPF LFs to staurosporine-induced apoptosis and responsiveness to transforming growth factor β(1) but increased basal α-SMA and restored β(3) integrin expression in LFs via an ALK-5-dependent, SMAD3/7-independent mechanism. These data suggest that STAT3 activation regulates several pathways in human LFs associated with normal wound healing, whereas aberrant STAT3 signaling plays a critical role in UIP/IPF pathogenesis.

A Puff of Air Sorts Bioaerosols for Pathogen Identification

Optical-based, real-time bioaerosol sensors are able to distinguish bioaerosols and nonbioaerosols but are unable to classify pathogens or even discriminate harmful from harmless bacteria. Biochemical techniques can identify genus and species of bacteria but cannot do so efficiently due to time limitation and the very high concentration of background aerosols. We report for the first time an efficient aerosol sorter consisting of an aerodynamic deflector that is cued from bioaerosol fluorescence. Bacillus subtilis (anthrax stimulant) aerosol was successfully separated from mixed aerosols. We achieved enrichment of the suspected bioaerosol concentration by at least 103, thus leading us new perspectives in pathogen identification. Although, at the present stage, a complete characterization of bioaerosols in real time is not yet possible, this new early warning/alarm sensor can supply high concentration of suspect bioaerosol particles for further specific analysis via bio-chemical assay technology or other subtle optical methods such as Raman spectroscopy.

Expression and activation of STAT proteins during mouse retina development.

Cytokines and growth factors play important roles in mammalian ocular development and maintenance. Recent studies have indicated that some of these ligands can activate signal transducer and activator of transcription factors (STATs) and modulate gene transcription. The purpose of this study was to investigate the expression and activation of STAT proteins in the developing mouse retina. Anti-STAT and anti-phosphorylated STAT antibodies were used to detect the expression and activation of STATs in embryonic and postnatal neuronal retina, ciliary margin, and retinal pigment epithelium (RPE). In situ hybridization and Western blot were also employed. In embryonic stages, all STAT proteins were expressed in the neuronal retina in distinct cell populations at different embryonic stages. For example, Stat3 expression and activation gradually increased in the inner neuroblast layer and ciliary margin during development. In adult retina, Stat3 was detected in the inner nuclear layer and ganglion cells layers. Stat1 was strongly expressed in both outer and inner plexiform layers. Stat5a was clearly expressed in the outer/inner nuclear layer, the ganglion cell layer, and the inner plexiform layer. Strong expression of Stat3, Stat5a, and Stat6 was observed in the RPE. Activated Stat3 and Stat5a were found in the neural retina and the RPE. Distinct STAT proteins were present in different cell populations in neuronal retina and RPE suggesting multiple functions of STATs in mammalian eye development. Studies of STAT signal pathways in the eye may contribute to the understanding of molecular mechanisms in control of ocular development and pathogenesis.

Molecular aspects of vertebrate retinal development.

The formation of retina from neural plate has been mapped extensively by anatomical and molecular methods. The major cascades of transcription factor expression have been identified, and deficits resulting from transcription factor knockouts are well characterized. There is extensive cross-regulation, both positive and negative, at the transcriptional level between transcription factors and this is vital in the formation of neural compartments. Many transcription factors are important at both early stages of optic cup formation and later stages of terminal differentiation of retinal cell types. The transcription factor cascades can be regulated by extrinsic factors, and some of the intracellular signaling pathways whereby this is achieved have been identified. Defining the quantitative interactions between regulatory molecules will be the next step in understanding this excellent model of vertebrate central nervous system (CNS) development.

DNA repair and cancer: Lessons from mutant mouse models

DNA damage, if the repair process, especially nucleotide excision repair (NER), is compromised or the lesion is repaired by some other error‐prone mechanism, causes mutation and ultimately contributes to neoplastic transformation. Impairment of components of the DNA damage response pathway (e.g., p53) is also implicated in carcinogenesis. We currently have considerable knowledge of the role of DNA repair genes as tumor suppressors, both clinically and experimentally. The deleterious clinical consequences of inherited defects in DNA repair system are apparent from several human cancer predisposition syndromes (e.g., NER‐compromised xeroderma pigmentosum [XP] and p53‐deficient Li‐Fraumeni syndrome). However, experimental studies to support the clinical evidence are hampered by the lack of powerful animal models. Here, we review in vivo experimental data suggesting the protective function of DNA repair machinery in chemical carcinogenesis. We specifically focus on the three DNA repair genes, O6‐methylguanine‐DNA methyltransferase gene (MGMT), XP group A gene (XPA) and p53. First, mice overexpressing MGMT display substantial resistance to nitrosamine‐induced hepatocarcinogenesis. In addition, a reduction of spontaneous liver tumors and longer survival times were evident. However, there are no known mutations in the human MGMT and therefore no associated cancer syndrome. Secondly, XPA mutant mice are indeed prone to spontaneous and carcinogen‐induced tumorigenesis in internal organs (which are not exposed to sunlight). The concomitant loss of p53 resulted in accelerated onset of carcinogenesis. Finally, p53 null mice are predisposed to brain tumors upon transplacental exposure to a carcinogen. Accumulated evidence in these three mutant mouse models firmly supports the notion that the DNA repair system is vital for protection against cancer.

Serum cytokine alteration is associated with optic neuropathy in human primary open angle glaucoma.

PurposeThe purpose of this study is to compare human serum levels of TH1 and TH2 cytokines between 2 stages of primary open-angle glaucoma (POAG) and nonglaucomatous controls. MethodsThirty-two patients with primary POAG and 26 normal control subjects were enrolled into this study. The 32 patients with POAG were divided into 2 subgroups according to their mean defect (MD) with MD better than −12 dB and worse than −12 dB on the visual field. Enzyme-linked immunosorbent assay was used to assay for the levels of TH1 cytokines serum soluble interleukin-2 receptor (sIL-2R), interleukin (IL)-2, IL-12p40, IL-12p70, IL-23, tumor necrosis factor (TNF)-alpha, interferon-gamma, and TH2 cytokines IL-4, IL-6 in the peripheral serum. ResultsPatients with mild glaucomatous neuropathy exhibited significant elevations in IL-4 (P<0.0001) and IL-6 (P=0.0302) compared with the controls, whereas higher concentrations of IL-4 (P<0.0001) and IL-6 (P=0.0084) were found in patients with severe glaucomatous neuropathy. The level of IL-12p70 was significantly increased in both the MD ≥12 dB (P<0.0001) and MD <12 dB (P<0.0001) groups. A significant decrease in TNF-alpha levels were observed in MD <12 dB group compared with the controls (P=0.0464), and TNF-alpha levels in MD <12 dB group were lower than MD ≥12 dB group (P=0.0328). No significant differences in serum concentrations of IL-2, sIL-2R IL-12p40, IL-23 and interferon-gamma were found between MD <12 dB group, MD ≥12 dB group, and control group. ConclusionsSignificant alterations of serum TH1 and TH2 cytokines are associated with glaucoma, suggesting the possibility that abnormal immune environments contribute to the glaucomatous neuropathy of POAG.

STAT3 activation protects retinal ganglion cell layer neurons in response to stress.

STAT3 is a major signaling molecule for many neurotrophic factors but its direct role in the protection of neurons in response to stress has not been addressed. We have studied the role of STAT3 in protecting retinal neurons from damage induced by ischemia/reperfusion and glutamate excitotoxicity by using adenovirus constructs to introduce active, normal or inactive STAT3 into retinal ganglion cells in culture and cells of the ganglion cell layer in the intact retina. Transient ischemia/reperfusion was induced in adult CD1 mice by elevating the intraocular pressure to the equivalent of 120mmHg for 60min, followed by a return to normal pressure. The levels, activation and distribution of STAT3 protein were evaluated by Western blot and immunocytochemistry. A transient peak of STAT3 activation was seen at 24h post ischemia and a strong increase in STAT3 protein levels 24h later. The increase in levels of STAT3 was detected in both ganglion cell bodies and processes in the plexiform layers by immunocytochemistry. The time course of STAT3 increase was slower than the time course of ganglion cell death as measured by TUNEL assay. Intravitreal injection of NMDA led to peak increases in activated STAT3 and STAT3 at 12 and 24h post insult respectively. Purified RGCs were infected with recombinant wild-type STAT3, constitutively active and dominant negative forms of STAT3 adenoviruses or control empty virus and then treated with glutamate. Surviving infected cells were counted 24 and 48h later. Infection with constitutively active STAT3 gave substantial protection when compared to the other constructs. Similarly, intravitreal injection of constitutively active STAT3 adenovirus one day before ischemia-reperfusion resulted in a decreased neural cell death in the ganglion cell layer compared with GFP adenovirus control. Our results suggest that persistent activation of STAT3 by neurotrophic factors provides strong neuroprotection and will be an effective strategy in a number of chronic retinal diseases.

Stage and Gene Specific Signatures Defined by Histones H3K4me2 and H3K27me3 Accompany Mammalian Retina Maturation In Vivo

The epigenetic contribution to neurogenesis is largely unknown. There is, however, growing evidence that posttranslational modification of histones is a dynamic process that shows many correlations with gene expression. Here we have followed the genome-wide distribution of two important histone H3 modifications, H3K4me2 and H3K27me3 during late mouse retina development. The retina provides an ideal model for these studies because of its well-characterized structure and development and also the extensive studies of the retinal transcriptome and its development. We found that a group of genes expressed only in mature rod photoreceptors have a unique signature consisting of de-novo accumulation of H3K4me2, both at the transcription start site (TSS) and over the whole gene, that correlates with the increase in transcription, but no accumulation of H3K27me3 at any stage. By in silico analysis of this unique signature we have identified a larger group of genes that may be selectively expressed in mature rod photoreceptors. We also found that the distribution of H3K4me2 and H3K27me3 on the genes widely expressed is not always associated with their transcriptional levels. Different histone signatures for retinal genes with the same gene expression pattern suggest the diversities of epigenetic regulation. Genes without H3K4me2 and H3K27me3 accumulation at any stage represent a large group of transcripts never expressed in retina. The epigenetic signatures defined by H3K4me2 and H3K27me3 can distinguish cell-type specific genes from widespread transcripts and may be reflective of cell specificity during retina maturation. In addition to the developmental patterns seen in wild type retina, the dramatic changes of histone modification in the retinas of mutant animals lacking rod photoreceptors provide a tool to study the epigenetic changes in other cell types and thus describe a broad range of epigenetic events in a solid tissue in vivo.