Direct activation of forkhead box O3 by tumor suppressors p53 and p73 is disrupted during liver regeneration in mice.

UNLABELLED: The p53 family of proteins regulates the expression of target genes that promote cell cycle arrest and apoptosis, which may be linked to cellular growth control as well as tumor suppression. Within the p53 family, p53 and the transactivating p73 isoform (TA-p73) have hepatic-specific functions in development and tumor suppression. Here, we determined TA-p73 interactions with chromatin in the adult mouse liver and found forkhead box O3 (Foxo3) to be one of 158 gene targets. Global profiling of hepatic gene expression in the regenerating liver versus the quiescent liver revealed specific, functional categories of genes regulated over the time of regeneration. Foxo3 is the most responsive gene among transcription factors with altered expression during regenerative cellular proliferation. p53 and TA-p73 bind a Foxo3 p53 response element (p53RE) and maintain active expression in the quiescent liver. During regeneration of the liver, the binding of p53 and TA-p73, the recruitment of acetyltransferase p300, and the active chromatin structure of Foxo3 are disrupted along with a loss of Foxo3 expression. In agreement with the loss of Foxo3 transcriptional activation, a decrease in histone activation marks (dimethylated histone H3 at lysine 4, acetylated histone H3 at lysine 14, and acetylated H4) at the Foxo3 p53RE was detected after partial hepatectomy in mice. These parameters of Foxo3 regulation are reestablished with the completion of liver growth and regeneration and support a temporary suspension of p53 and TA-p73 regulatory functions in normal cells during tissue regeneration. p53-dependent and TA-p73-dependent activation of Foxo3 was also observed in mouse embryonic fibroblasts and in mouse hepatoma cells overexpressing p53, TA-p73alpha, and TA-p73beta isoforms. CONCLUSION: p53 and p73 directly bind and activate the expression of the Foxo3 gene in the adult mouse liver and murine cell lines. p53, TA-p73, and p300 binding and Foxo3 expression decrease during liver regeneration, and this suggests a critical growth control mechanism mediated by these transcription factors in vivo.

Elevated interferon gamma signaling contributes to impaired regeneration in the aged liver.

Our previous study on immune-related changes in the aged liver described immune cell infiltration and elevation of inflammation with age. Levels of interferon (IFN)-γ, a known cell cycle inhibitor, were elevated in the aging liver. Here, we determine the role played by IFN-γ in the delayed regenerative response observed in the aged livers. We observed elevated IFN signaling in both aged hepatocytes and regenerating livers post-partial hepatectomy. In vivo deletion of the major IFN-γ producers-the macrophages and the natural killer cells, leads to a reduction in the IFN-γ levels accompanied with the restoration of the DNA synthesis kinetics in the aged livers. Eighteen-month-old IFN-γ-/- mice livers, upon resection, exhibited an earlier entry into the cell cycle compared with age-matched controls. Thus, our study strongly suggests that an age-related elevation in inflammatory conditions in the liver often dubbed as "inflammaging" has a detrimental effect on the regenerative response.

C/EBPalpha triggers proteasome-dependent degradation of cdk4 during growth arrest.

CCAAT/enhancer binding protein alpha (C/EBPalpha) causes growth arrest via direct interaction with the cyclin-dependent kinases cdk2 and cdk4. In this paper, we present evidence showing that C/EBPalpha enhances a proteasome-dependent degradation of cdk4 during growth arrest in liver of newborn mice and in cultured cells. Overexpression of C/EBPalpha in several biological systems leads to a reduction of cdk4 protein levels, but not mRNA levels. Experiments with several tissue culture models reveal that C/EBPalpha enhances the formation of cdk4-ubiquitin conjugates and induces degradation of cdk4 through a proteasome-dependent pathway. As a result, the half-life of cdk4 is shorter and protein levels of cdk4 are reduced in cells expressing C/EBPalpha. Gel filtration analysis of cdk4 complexes shows that a chaperone complex cdk4-cdc37-Hsp90, which protects cdk4 from degradation, is abundant in proliferating livers that lack C/EBPalpha, but this complex is weak or undetectable in livers expressing C/EBPalpha. Our studies show that C/EBPalpha disrupts the cdk4-cdc37-Hsp90 complex via direct interaction with cdk4 and reduces protein levels of cdk4 by increasing proteasome-dependent degradation of cdk4.

The role of substance P in inflammatory disease.

The diffuse neuroendocrine system consists of specialised endocrine cells and peptidergic nerves and is present in all organs of the body. Substance P (SP) is secreted by nerves and inflammatory cells such as macrophages, eosinophils, lymphocytes, and dendritic cells and acts by binding to the neurokinin-1 receptor (NK-1R). SP has proinflammatory effects in immune and epithelial cells and participates in inflammatory diseases of the respiratory, gastrointestinal, and musculoskeletal systems. Many substances induce neuropeptide release from sensory nerves in the lung, including allergen, histamine, prostaglandins, and leukotrienes. Patients with asthma are hyperresponsive to SP and NK-1R expression is increased in their bronchi. Neurogenic inflammation also participates in virus-associated respiratory infection, non-productive cough, allergic rhinitis, and sarcoidosis. SP regulates smooth muscle contractility, epithelial ion transport, vascular permeability, and immune function in the gastrointestinal tract. Elevated levels of SP and upregulated NK-1R expression have been reported in the rectum and colon of patients with inflammatory bowel disease (IBD), and correlate with disease activity. Increased levels of SP are found in the synovial fluid and serum of patients with rheumatoid arthritis (RA) and NK-1R mRNA is upregulated in RA synoviocytes. Glucocorticoids may attenuate neurogenic inflammation by decreasing NK-1R expression in epithelial and inflammatory cells and increasing production of neutral endopeptidase (NEP), an enzyme that degrades SP. Preventing the proinflammatory effects of SP using tachykinin receptor antagonists may have therapeutic potential in inflammatory diseases such as asthma, sarcoidosis, chronic bronchitis, IBD, and RA. In this paper, we review the role that SP plays in inflammatory disease.

Upregulation of neurokinin-1 receptor expression in the lungs of patients with sarcoidosis.

Substance P (SP) is a proinflammatory neuropeptide that is secreted by sensory nerves and inflammatory cells. Increased levels of SP are found in sarcoid bronchoalveolar lavage fluid. SP acts by binding to the neurokinin-1 receptor and increases secretion of tumor necrosis factor-alpha in many cell types. We sought to determine neurokinin-1 receptor expression in patients with sarcoidosis compared with normal controls. Neurokinin-1 receptor messenger RNA and protein expression were below the limits of detection by reverse transcriptase-polymerase chain reaction and immunohistochemistry in peripheral blood mononuclear cells of healthy volunteers (n = 9) or patients with stage 1 or 2 pulmonary sarcoidosis (n = 10), but were detected in 1/9 bronchoalveolar lavage cells of controls compared with 8/10 patients with sarcoidosis (p = 0.012) and 2/9 biopsies of controls compared with 9/10 patients with sarcoidosis (p = 0.013). Immunohistochemistry localized upregulated neurokinin-1 receptor expression to bronchial and alveolar epithelial cells, macrophages, lymphocytes, and sarcoid granulomas. The patient in whom neurokinin-1 receptor was not detected was taking corticosteroids. Incubation of the type II alveolar and bronchial epithelial cell lines A549 and SK-LU 1 with dexamethasone downregulated neurokinin-1 receptor expression. Upregulated neurokinin-1 receptor expression in patients with sarcoidosis may potentiate substance P-induced proinflammatory cytokine production in patients with sarcoidosis.

Neurokinin-1 receptor (NK-1R) expression is induced in human colonic epithelial cells by proinflammatory cytokines and mediates proliferation in response to substance P.

We have previously shown that the receptor for substance P (SP), neurokinin-1 receptor (NK-1R), is a marker of human mucosal but not peripheral mononuclear cells. In the present study, we investigate NK-1R expression in the human colonic mucosa in vivo, particularly in the epithelial cells. We investigate the influence of proinflammatory Th1 cytokines and SP on expression and function of NK-1R in colonic epithelial cells in vitro. Using in situ hybridization to detect NK-1R mRNA, and immunohistochemistry to detect NK-1R protein, colonic epithelial cells were found to express NK-1R in vivo. In contrast, colon epithelial cell lines (Caco-2, HT29, SW620, T84) were negative for NK-1R mRNA and protein. However, stimulation with a proinflammatory cytokine cocktail containing IFN-gamma, TNF-alpha, and IL-1beta, caused induction of NK-1R expression. Expression of NK-1R in human colonic epithelial cells in vivo may therefore reflect cytokine conditioning by the mucosal microenvironment. SP did not alter ion transport in monolayers of cytokine-treated T84 cells. While SP stimulated epithelial ion transport in colonic mucosae ex vivo, this was not a direct effect of SP on the epithelial cells, and appeared to be neurally mediated. However, SP (10(-10)-10(-8) M) elicited a dose-dependent proliferative effect on cytokine-stimulated, but not unstimulated, SW620 cells. Proliferation of the epithelial cells in response to SP was mediated specifically via cytokine-induced NK-1R, since an NK-1R-specific antagonist (Spantide 1) completely blocked SP-mediated proliferation in the cytokine-treated cells. Our results therefore demonstrate that proinflammatory cytokines induce expression of NK-1R in human colonic epithelial cell lines, and that SP induces proliferation of the epithelial cells via cytokine-induced NK-1R.