Gross Cystic Disease Fluid Protein-15(GCDFP-15)/Prolactin-Inducible Protein (PIP) as Functional Salivary Biomarker for Primary Sjögren's Syndrome

Abstract

Background: Gross cystic disease fluid protein-15(GCDFP-15)/prolactin-inducible protein (PIP) is a secretory acinar glycoprotein of 14 KDa which we have recently described as significantly lower in salivary samples of patients with primary Sjögren's syndrome (pSS) in comparison to healthy volunteers by proteomic analysis.

Aims of the study: (1) to validate our previous data on the decrease of GCDFP-15/PIP protein in a larger number of subjects with pSS (2) to integrate the proteomic results with complementary immunoassays in order better clarify the pathophysiological relevance of GCDFP-15/PIP in pSS exocrinopathy (3) to assess both the glandular expression of the GCDFP-15/PIP and the levels of glandular GCDFP-15/PIP mRNA in the patients' minor salivary gland (MSG) biopsies in order to verify whether the observed reduction of GCDFP-15/PIP in saliva may be related to a decrease in the protein production.

Patients and methods: A total of 123 salivary samples from patients affected by pSS, no-SS sicca syndrome and sex- age-matched healthy volunteers were analyzed by different proteomic techniques (SELDI-TOF-MS, 2DE, MALDI-TOF-MS). The expression of GCDFP-15/PIP was then validated by western blot analysis. Real Time PCR and immunohistochemistry for GCDFP-15/PIP in the minor salivary glands (MSG) biopsies were then carried out.

Results: By using complementary proteomic analysis we found that a putative peak of 16547 m/z was among the best independent biomarkers for pSS able to discriminate between patients and healthy controls with a sensitivity of 96 % and a specificity of 70%, with a global cross validated error of 29%. We identified the peak as the GCDFP-15/PIP protein and verified that the intensity of GCDFP-15/PIP was significantly lower in pSS patients when compared to both no-SS sicca subjects and healthy controls (p<0.0001). GCDFP-15/PIP expression also correlated with both the salivary flow rate (r=0.312, p=0.023) and MSG biopsies focus score (r=-0.377, p=0.04). Finally, immunohistochemistry confirmed that GCDFP-15/PIP staining was faint in mucus acini and Real Time PCR showed that GCDFP-15/PIP mRNA was significantly lower in pSS patients when compared to both no-SS sicca subjects and healthy controls (p=0.023) thus supporting the hypothesis that the observed reduction of GCDFP-15/PIP in pSS saliva may be related to a decrease in the protein production.

Conclusion: In this study by different complementary-omic techniques we confirmed the potential role of GCDFP-15/PIP as a novel biomarker for pSS. This finding might also be functionally important as GCDFP-15/PIP has previously been shown to bind to Aquaporin 5 (AQP5), a salivary gland water channel, critical to saliva formation that is known to be downregulated in pSS. It is likely that exploring the GCDFP-15/PIP/AQP5 axis will help better understand the mechanism of salivary gland dysfunction in pSS.

Keywords: Autoimmune disease; GCDFP-15/PIP; Sjören’s syndrome.

Figure 1

Flow chart of the patients’ enrolled in the study (2DE, 2D electrophoresis; WB, western blot; MSGB, minor salivary gland biopsies; IHC, immunohistochemistry).

Figure 2

Classification and regression tree (CART) algorithm, as result of the analysis of the SELDI-TOF-MS results (SS, Sjögren’s syndrome; HC, healthy control, V63=16547 m/z, V66=24059 m/z, V54=13507 m/z, V55=13714 m/z, V34=7149 m/z, V35=7192 m/z).

Figure 3

Representative 2DE - gel map from a) primary Sjögren’s syndrome patient b) healthy control.

Figure 4

Median and interquartile range of GCDFP-15/PIP optical density of patients and healthy controls (p<0.0001 by ANOVA analysis). GCDFP-15/PIP is differently expressed between primary Sjögren’s syndrome patients (pSS) and non-SS sicca syndrome patients (CTL), with p=0.01 (*), and between pSS and healthy control subjects (HC), with p<0.0001 (***).

Figure 5

Representative Western blot comparative analysis between Sjögren’s syndrome patients (pSS), non-SS sicca syndrome patients (CTL), and healthy control subjects (HC). a) acquisition of total protein content run during 1D SDS-electrophoresis, after the stain-free gel was UV-activated; b) acquisition of immunoreactive GCDFP-15/PIP band on a nitrocellulose film.

Figure 6

Difference of salivary flow rate between patients with low versus normal GCDFP-15/PIP expression, as result of a qualitative western blot analysis, obtained by using the minimum value of GCDFP-15/PIP expression level of healthy controls as a cut-off point to discriminate between a low and a normal expression (Mann Whitney test, p=0.0022).

Figure 7

Difference of mRNA expression (GCDFP-15/PIP mRNA expression normalized on GAPDH) between patients with primary Sjögren’s syndrome (pSS) and no-SS sicca syndrome subjects (CTL) (Mann Whitney test, p=0.023).

Figure 8

Immunohistochemical studies of GCDFP-15/PIP in sections derived from SS biopsies and healthy volunteer. In healthy volunteer the staining is more concentrated towards the apical pole of mucuse cells (acinous marked with m in figure 8A’) and ductal cell are negative, is also possible observe some immunorective material in the lumen of mayor ducts (asterisk in figure 8A). In SS patients the immunoreactivity for GCDFP-15/PIP is faint in mucus acini (see m in figure 8B’), no major changes were observed in serous acinar cells but slightly increase in the intensity of the immunoreactivity in this cells.