Transcription factors Runx1 to 3 are expressed in the lacrimal gland epithelium and are involved in regulation of gland morphogenesis and regeneration

Abstract

Purpose: Lacrimal gland (LG) morphogenesis and repair are regulated by a complex interplay of intrinsic factors (e.g., transcription factors) and extrinsic signals (e.g., soluble growth/signaling factors). Many of these interconnections remain poorly characterized. Runt-related (Runx) factors belong to a small family of heterodimeric transcription factors known to regulate lineage-specific proliferation and differentiation of stem cells. The purpose of this study was to define the expression pattern and the role of Runx proteins in LG development and regeneration.

Methods: Expression of epithelial-restricted transcription factors in murine LG was examined using immunostaining, qRT-PCR, and RT(2)Profiler PCR microarrays. The role of Runx transcription factors in LG morphogenesis was studied using siRNA and ex vivo LG cultures. Expression of Runx transcription factors during LG regeneration was assessed using in vivo model of LG regeneration.

Results: We found that Runx factors are expressed in the epithelial compartment of the LG; in particular, Runx1 was restricted to the epithelium with highest level of expression in ductal and centroacinar cells. Downregulation of Runx1 to 3 expression using Runx-specific siRNAs abolished LG growth and branching and our data suggest that Runx1, 2, and 3 are partially redundant in LG development. In siRNA-treated LG, reduction of branching correlated with reduction of epithelial proliferation, as well as expression of cyclin D1 and the putative epithelial progenitor cell marker cytokeratin-5. Runx1, Runx3, and cytokeratin-5 expression increased significantly in regenerating LG and there was modest increase in Runx2 expression during LG differentiation.

Conclusions: Runx1 and 2 are new markers of the LG epithelial lineage and Runx factors are important for normal LG morphogenesis and regeneration.

Keywords: Runx1; Runx2; Runx3; lacrimal gland regeneration; qRT PCR microarrays; stem and progenitor cells.

Figure 1

Stem cell PCR array revealed genes differentially expressed in LG epithelium and mesenchyme. Total RNA was prepared from E16.6 enzymatically separated LG epithelium and mesenchyme. Relative expression was calculated using the household genes provided in the array. Bars represent means ± SEM of two experiments. LG epithelium was enriched with expression of collagen, type II, alpha 1 (Col2a1), keratin 15 (Krt5), cadherin 1 (Cdh1), fibroblast growth factor 1 (Fgf1), gap junction protein, beta 1 (Gjb1), Cyclin D1 (CcnD1), delta-like 1 (Dll1), myelocytomatosis oncogene (Myc), aldehyde dehydrogenase family 1, subfamily A1 (Aldh1a1), membrane metalloendopeptidase (Mme), cell division cycle 42 homolog (Cdc42), Cyclin A2 (Ccna2). LG mesenchyme was enriched with expression of peroxisome proliferator activated receptor gamma (Pparg), fibroblast growth factor 2 (Fgf2), insulin-like growth factor 1 (Igf1), chemokine (C-X-C motif) ligand 12 (Cxcl12), S100 protein, beta polypeptide (S100b), CD44 antigen (CD44), aldehyde dehydrogenase family 1, subfamily A1 (Aldh1a1), bone morphogenetic protein 3 (Bmp3), aggrecan (Acan), CD4 antigen (CD4), bone morphogenetic protein 2 (Bmp2), ATP-binding cassette, subfamily G (WHITE), member 2 (Abcg2), delta-like 3, (Dll3), CD3 antigen, delta polypeptide (CD3d), desert hedgehog (Dhh), actin, alpha, cardiac muscle 1 (Actc1), CD8 antigen, alpha chain (CD8a).

Figure 2

Analysis of Runx1 expression in the lacrimal gland of heterozygous Runx1 knockin-lacZ mice. (A, B) Whole-mount X-Gal staining of mouse embryo at E17.0. Runx1 expression is restricted to the epithelium of the LG and PG; (B) shows an excised LG. Red arrows mark epithelial component of the lacrimal gland. (C) Runx1-lacZ is expressed in the SLG and SMG. (D, E) Runx1 is also expressed in the epithelial and mesenchymal components of the meibomian gland (mei) and lung epithelium (F). (G–L) Expression pattern of Runx1 in the LGs at postnatal development day 0 (P0; [G, H]), P17 (I, J), 6-month-old (K, L) old mice. Runx1 expression is restricted to the ducts and centroacinar cells (black arrows) within the secretory acini. LGs obtained from Runx1 knockin-lacZ mice were stained with X-Gal and paraffin sections were prepared. Nuclei were stained with fast red to visualize LG morphology. (M–O) PAS reaction was also used for staining of sections of Runx1-LacZ LGs. Strong LacZ staining was seen within all PAS⁺ ducts, including a main duct (m. duct) and centroacinar cells (yellow arrows) and within some PAS^– acini/cells (white arrows).

Figure 3

Mutation in the Runx1 gene reduces LG budding and perturbs HG development in embryos at E16.5. (A–C) LG; (D–F) HG development in WT (Runx+/+) (A, D) and mutant (Runx–/–) (B, C, E, F) embryos. Yellow arrows indicate LG budding, white arrow conjunctiva, and red arrow HG epithelium component in WT embryos.

Figure 4

Knockdown of Runx1, 2, and 3 reduces LG epithelial growth, cell proliferation, and number of Krt5-expressing progenitor cells. (A) Scheme of ex vivo siRNA treatment. LG were isolated at one bud stage (also see [C]) at E15.5 and cultured on a filter floating on the surface of culture medium. Control and Runx siRNAs were delivered using RNAiMAX (Invitrogen). (B) Runx1, 2, and 3 RNAs fold decrease after specific siRNA application is shown relative to control-treated LGs. (C) Example of LG at one bud stage used for siRNA application. (D) CY-3–labeled control RNA is found in all cells of the LG. (E) Scrambled control siRNA application does not affect LG branching. (F) Combined specific Runx1, 2, and 3 siRNA applications strongly reduce LG growth/branching. (G) Quantification of buds in siRNA-treated glands shows significant inhibition of branching after application of Runx1, or combinations of Runx1 and 2 or Runx1, 2, and 3 siRNAs (E-cadherin is green, PECAM is red). (H–K) The number of proliferating cells is decreased in Runx-siRNA treated LGs (H) quantification of H-3P antibody-stained proliferating cells in control and Runx1, 2, and 3 siRNA-treated LG buds. Control (I) and Runx1, 2, and 3 siRNA-treated (J–K) LG were stained with H-3P antibody (green), E-cadherin (red), and 4′,6-diamidino-2-phenylindole (blue) (white arrowheads show epithelium). LGs treated with Runx1, 2, and 3 siRNA show almost no cell proliferation in the epithelial component of the gland, while proliferating cells are still present in mesenchyme. (L) Number of Krt5-expressing cells is strongly reduced in LGs treated with Runx1, 2, and 3 siRNA. Asterisks denote statistical significance in the results compared to the control.

Figure 5

Knockdown of Runx1, 2, and 3 expression results in decreased CyclinD1 protein. (A) Protein was prepared from siRNA-treated LG organ cultures, resolved by SDS-PAGE, and probed with the cyclin-D1 or β-actin antibodies. (B) Graphic representation of densitometric analyses of Western blots from two independent experiments (mean ± SD of Optical Density [OD] of bands). For analysis of the Western blot images ImageJ 1.46 (National Institutes of Health, Bethesda, MD) has been used. The relative densities of the loading-control bands and the sample bands were calculated. Adjusted density for each sample lane was calculated by dividing the sample relative density by the loading-control relative density for each lane separately. Asterisks mark significant statistical difference in the level of CyclinD1 of experimental siRNA treated lacrimal glands compared to control treated glands.

Figure 6

Graphic representation of Table 5. LGs were injected with IL-1 or saline (vehicle control) and collected at day 1, 2, 3, 7, or 21 after injection for RNA extraction. Levels of Runx1, Runx2, Runx3, Krt5, RBM3, and PrL27 and PrS13 mRNAs were determined by qRT-PCR. Increase in Runx1 and Runx3 expression correlates with increase in expression of epithelial progenitor cell marker Krt5, while expression of Runx2 and ubiquitously expressed Rbm3 remains unchanged during regeneration.