Increased expression and cellular localization of spermine oxidase in ulcerative colitis and relationship to disease activity

Abstract

Background: Polyamines are important in cell growth and wound repair, but have also been implicated in inflammation-induced carcinogenesis. Polyamine metabolism includes back-conversion of spermine to spermidine by the enzyme spermine oxidase (SMO), which produces hydrogen peroxide that causes oxidative stress. In ulcerative colitis (UC), levels of spermine are decreased compared to spermidine. Therefore, we sought to determine if SMO is involved in UC.

Methods: Colon biopsies and clinical information from subjects undergoing colonoscopy for evaluation of UC or colorectal cancer screening were utilized from 16 normal controls and 53 UC cases. Histopathologic disease severity was graded and the Mayo Disease Activity Index (DAI) and endoscopy subscore assessed. SMO mRNA expression was measured in frozen biopsies by TaqMan-based real-time polymerase chain reaction (PCR). Formalin-fixed tissues were used for SMO immunohistochemistry.

Results: There was a 3.1-fold upregulation of SMO mRNA levels in UC patients compared to controls (P = 0.044), and a 3.7-fold increase in involved left colon versus paired uninvolved right colon (P < 0.001). With worsening histologic injury in UC there was a progressive increase in SMO staining of mononuclear inflammatory cells. There was a similar increase in SMO staining with worsening endoscopic disease severity and strong correlation with the DAI (r = 0.653, P < 0.001). Inflammatory cell SMO staining was increased in involved left colon versus uninvolved right colon.

Conclusions: SMO expression is upregulated in UC tissues, deriving from increased levels in mononuclear inflammatory cells. Dysregulated polyamine homeostasis may contribute to chronic UC by altering immune responses and increasing oxidative stress.

Figures

Figure 1

SMO mRNA expression in colon tissues from normal controls and patients with UC. Gene expression was assessed by real-time PCR using TaqMan-based multiplex PCR with standardization of levels to the housekeeping gene GAPDH. When all patients with UC were considered, there was a significant increase in SMO mRNA levels (P = 0.044 by unpaired Student’s t-test).

Figure 2

Comparison of SMO mRNA levels in UC-involved left-sided colon tissues with uninvolved right colon from the same patients determined by real-time PCR using Taq-Man-based multiplex PCR. There was a significant increase in SMO mRNA levels from uninvolved right colon to involved left colon (P < 0.001 by paired Student’s t-test).

Figure 3

SMO protein expression by immunohistochemistry in subjects with normal histology and increasing grades of colitis as determined by histopathology. (A) Representative SMO immunoperoxidase staining observed in normal mucosa and in quiescent, mild, moderate, and severe UC tissues. The histopathologic disease status and microscopic magnification are shown. Note the progressively increased staining intensity of the inflammatory cells in the lamina propria of the UC samples with increasing disease activity, and that this staining is in mononuclear cells, with absence of staining of polymorphonuclear cells infiltrating the epithelial surface. (B) Quantification of immunohistochemistry staining score for SMO in colonic epithelium for the 5 groups of subjects (normal, and quiescent, mild, moderate, or severe UC). Overall P-value was 0.07 by ANOVA. (C) SMO expression in the inflammatory cells demonstrating that there was a significant increase in immunohistochemistry staining score with increasing disease severity by histopathology (overall P-value < 0.001 by ANOVA).

Figure 4

Comparison of SMO inflammatory cell protein expression levels as assessed by immunohistochemistry, with clinical parameters. (A) SMO protein levels by endoscopic disease category. Overall P-value < 0.001 by ANOVA. SMO levels were higher in the mild, moderate, and severe groups when compared to the cases with normal appearing mucosa. (B) Spline graph of SMO protein expression versus the DAI. Protein levels were strongly correlated with DAI, as determined by Pearson’s correlation test (r = 0.653, P < 0.0001).

Figure 5

Quantification of immunohistochemistry staining scores in 22 UC patient subjects, in which the right colon biopsies uninvolved with disease were compared to diseased left colon biopsies from the same patient, using a paired Student’s t-test. (A) There was no significant difference (P = 0.49) in SMO staining score in the comparison of the diseased left colon to the normal right colon in the epithelial cell component. (B) Increased SMO staining score in the lamina propria mononuclear cells (P = 0.01) in the diseased left colon in comparison to the normal right colon.