The Matricellular Protein SPARC Decreases in the Lacrimal Gland At Adulthood and During Inflammation

Abstract

Purpose: Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein abundantly expressed in basement membranes and capsules surrounding a variety of organs and tissues. It mediates extracellular matrix organization and has been implicated in cell contraction. Here, we evaluated the expression of SPARC in the murine lacrimal gland at adulthood and during inflammation.

Methods: Lacrimal glands of young mice (4-6 weeks old) and adult mice (32-40 weeks old) were used for extraction of DNA, RNA, and protein. The presence of SPARC was assessed by quantitative PCR, ELISA, and immunofluorescence microscopy. 5-Methylcytosine and DNA methylation were evaluated using ELISA and bisulfite genomic sequencing, respectively. The effects of cytokines and inflammation in Sparc expression were evaluated in vitro and in the non-obese diabetic (NOD) mouse model of Sjögren's syndrome.

Results: The mRNA and protein levels of SPARC were downregulated in lacrimal glands of mature adult mice presenting age-related histological alterations such as increased deposition of lipofuscin and lipids. Epigenetic analyses indicated that glands in adult mice contain higher levels of global DNA methylation and show increased hypermethylation of specific CpG sites within the Sparc gene promoter. Analysis of smooth muscle actin (SMA)-green fluorescent protein (GFP) transgenic mice revealed that SPARC localizes primarily to myoepithelial cells within the gland. Treatment of myoepithelial cells with IL-1β or TNF-α and the development of inflammation in the NOD mice led to decreased transcription of Sparc.

Conclusions: SPARC is a novel matricellular glycoprotein expressed by myoepithelial cells in the lacrimal gland. Loss of SPARC during adulthood and chronic inflammation might have detrimental consequences on myoepithelial cell contraction and the secretion of tear fluid.

Figure 1.

Age-related histological alterations in lacrimal glands of adult mice. (A) H&E staining of the exorbital lacrimal gland in C57BL/6J mice. The magnified area shows acini formed by epithelial cells and their nuclei. (B) Carbol fuchsin and Oil Red O staining of lacrimal gland cryosections from young (4–6 weeks old) and adult (32–40 weeks old) C57BL/6J mice. Scale bars: 100 µm (zoomed-in portion, 25 µm).

Figure 2.

SPARC is downregulated in the adult lacrimal gland. (A) Total RNA was isolated from exorbital lacrimal glands from C57BL/6J mice at different ages (4–6 weeks old, 17–18 weeks old, and 32–40 weeks old; n = 5 or 6 mice per group). The relative levels of Sparc expression were determined using qPCR. (B) Total protein extracts were prepared from homogenized exorbital lacrimal glands from young (4–6 weeks old) and adult (32–40 weeks old) C57BL/6J mice and analyzed using ELISA (n = 8 mice/group). The box-and-whisker plots show the 25th and 75th percentiles (boxes), the median, and the minimum and maximum data values (whiskers). Significance was determined using the non-parametric Kruskal–Wallis test with Dunn's test for multiple comparisons (A) or the Mann–Whitney test (B). *P < 0.05; **P < 0.01.

Figure 3.

The levels of 5-mC increase in the adult lacrimal gland. (A) Diagrams depicting the methylation (red) of the DNA base cytosine (C) into 5-mC (above) and the Sparc promoter region with specific CpG sites (below). TSS, transcription start site. (Adapted from Tajerian et al.¹⁹) (B) The gDNA was isolated from exorbital lacrimal glands (n = 18 mice/group) and skin (n = 16 mice/group) from young (4–6 weeks old) and adult (32–40 weeks old) C57BL/6J mice. The levels of 5-mC were determined using ELISA. The box-and-whisker plots show the 25th and 75th percentiles (boxes), the median, and the minimum and maximum data values (whiskers). Significance was determined using the Mann–Whitney test. ***P < 0.001; ****P < 0.0001.

Figure 4.

Methylation state of CpG sites in the Sparc gene promoter region. The gDNA was isolated from exorbital lacrimal glands (n = 6 mice/group) from young (4–6 weeks old) and adult (32–40 weeks old) C57BL/6J mice. The methylation of four specific CpG sites in the Sparc gene promoter region (−208, −171, −135, and −101) was evaluated by bisulfite genomic sequencing after PCR amplification and cloning into a vector. The graph shows the percentage of methylated and unmethylated sites in each group of mice, as well as the total number of sequences analyzed (in parentheses).

Figure 5.

SPARC localizes to lacrimal gland myoepithelial cells. (A) Cryosections of lacrimal gland tissue from 4-week-old SMA–GFP mice were incubated with an anti-SPARC antibody. Merged images show colocalization of SPARC (red) with GFP-expressing myoepithelial cells (green). A fine punctate staining of SPARC can be observed within the extracellular space. (B) Immunostaining of SPARC (red) in cultured myoepithelial cells (green) isolated from lacrimal glands of 4- to 6-week-old SMA–GFP mice. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bars: 50 µm.

Figure 6.

Sparc transcription decreases during inflammatory conditions. (A) Total RNA was obtained from myoepithelial cells isolated from lacrimal glands of SMA–GFP mice. The cells were cultured with IL-1β, TNF-α, or vehicle control. The relative levels of Sparc expression were determined using qPCR (n = 3–6 independent experiments). (B) H&E staining of the exorbital lacrimal glands of control 16-week-old BALB/c mice and 16-week-old NOD mice. Lacrimal glands from BALB/c mice show normal morphology with lobules devoid of immune cells. In contrast, lacrimal glands from NOD mice are heavily infiltrated by immune cells that formed large foci within the lobules (arrows). Scale bar: 100 µm. Total RNA isolated from the lacrimal glands of these mice was used to determine the relative levels of Sparc expression using qPCR (n = 6 mice/group). The box-and-whisker plots show the 25th and 75th percentiles (boxes), the median, and the minimum and maximum data values (whiskers). Significance was determined using the non-parametric Kruskal–Wallis test with Dunn's test for multiple comparisons (A) or the Mann–Whitney test (B). *P < 0.05; **P < 0.01.