MUC1 expression in Sjogren's syndrome, KCS, and control subjects

Abstract

Purpose: To quantify and compare human mucin 1 (MUC1) protein and mRNA expression in tears and conjunctival epithelial cells collected from Sjogren's syndrome (SS), non-Sjogren's keratoconjunctivitus sicca (KCS) and non-dry eyed (NDE) control subjects.

Methods: Seventy-six subjects were recruited for this study: 25 SS (confirmed via American-European Consensus Criteria 2002), 25 KCS (confirmed by symptoms and Schirmer scores < or = 10 mm) and 26 NDE. Tears were collected using an eye-wash technique. Impression cytology was used to gather protein and mRNA from conjunctival epithelial cells. Soluble and membrane bound MUC1 were quantified via western blotting and MUC1 mRNA was quantified by real time qPCR.

Results: The SS group demonstrated significantly higher concentrations of soluble MUC1 (0.12 +/- 0.11 [SS]; 0.013 +/- 0.02 [KCS; p=0.001]; 0.0023 +/- 0.0024 [NDE; p<0.001]) and MUC1 mRNA (3.18 +/- 1.44 [SS]; 1.79 +/- 1.18 [KCS; p<0.05]; 1.60 +/- 0.74 [NDE; p<0.05]) compared to both KCS and NDE groups. Soluble MUC1 expression was also higher in the KCS group compared to the NDE group (p=0.02), where as MUC1 mRNA expression was similar in both KCS and NDE groups. Membrane bound MUC1 expression differed only between the SS and NDE groups (0.005 +/- -0.003 [SS]; 0.003 +/- 0.002 [NDE; p=0.002]).

Conclusions: These results demonstrate that SS subjects express greater quantities of MUC1 protein and mRNA compared to both KCS and control subjects. Increased soluble MUC1 expression was also found in KCS subjects compared to controls. Membrane bound MUC1 was present in higher concentration in SS versus NDE only. These significant changes in MUC1 expression may represent compensatory or protective responses to chronic insult to the ocular surface.

Figure 1

Western blot and regression analysis for soluble MUC1 quantification A: Examples of Soluble MUC1 western blots from tear samples derived from Normal, KCS, and SS and subjects. Lanes 1–6 are MUC1 standard antigen (CA15–3) Units; (Lane 1=0.3, Lane 2=0.1, Lane 3=0.06, Lane 4=0.03, Lane 5=0.01, Lane 6=0.003 U); Lanes 7–13 are examples of tear samples from Normal, Lanes 14–20 are from KCS, and Lanes 21–26 are from SS. B: A regression curve was created by graphing applied concentration of CA15–3 standard against the optical density of the resulting band immunoreactivity. Total MUC1 concentration was quantified by interpolation from this curve. MW standard (460) is listed on the left.

Figure 2

Soluble MUC1 expression as quantified by western blotting. Data expressed as (A) scatter graph of individual data points and (B) mean data. Protein samples collected via eye wash and MUC1 data expressed in Units/μg protein as calculated from interpolation from a standard curve titration of CA15–3. The asterisk indicates significantly different compared to NDE Group. The sharp (hash mark) indicates significantly different compared to KCS Group.

Figure 3

Expression of MUC1 mRNA in conjunctival epithelial cells. RNA isolated from conjunctival epithelial cells collected via impression cytology. The asterisk indicates significantly different compared to the NDE Group and the sharp (hash mark) indicates significantly different compared to the KCS Group.

Figure 4

Membrane bound MUC1 expression as quantified by western blotting. Protein samples collected via impression cytology. MUC1 data expressed in Units/μg protein as calculated from interpolation from a standard curve made from titration of CA15–3. The asterisk indicates significantly different compared to the NDE Group.