FOXC1 modulates MYOC secretion through regulation of the exocytic proteins RAB3GAP1, RAB3GAP2 and SNAP25

Abstract

The neurodegenerative disease glaucoma is one of the leading causes of blindness in the world. Glaucoma is characterized by progressive visual field loss caused by retinal ganglion cell (RGC) death. Both surgical glaucoma treatments and medications are available, however, they only halt glaucoma progression and are unable to reverse damage. Furthermore, many patients do not respond well to treatments. It is therefore important to better understand the mechanisms involved in glaucoma pathogenesis. Patients with Axenfeld-Rieger syndrome (ARS) offer important insight into glaucoma progression. ARS patients are at 50% risk of developing early onset glaucoma and respond poorly to treatments, even when surgical treatments are combined with medications. Mutations in the transcription factor FOXC1 cause ARS. Alterations in FOXC1 levels cause ocular malformations and disrupt stress response in ocular tissues, thereby contributing to glaucoma progression. In this study, using biochemical and molecular techniques, we show that FOXC1 regulates the expression of RAB3GAP1, RAB3GAP2 and SNAP25, three genes with central roles in both exocytosis and endocytosis, responsible for extracellular trafficking. FOXC1 positively regulates RAB3GAP1 and RAB3GAP2, while either increase or decrease in FOXC1 levels beyond its normal range results in decreased SNAP25. In addition, we found that FOXC1 regulation of RAB3GAP1, RAB3GAP2 and SNAP25 affects secretion of Myocilin (MYOC), a protein associated with juvenile onset glaucoma and steroid-induced glaucoma. The present work reveals that FOXC1 is an important regulator of exocytosis and establishes a new link between FOXC1 and MYOC-associated glaucoma.

Fig 1. Enrichment of FOXC1 at upstream regions of RAB3GAP1, RAB3GAP2 and SNAP25.

(A) Potential FOXC1 binding sites found upstream of RAB3GAP1, RAB3GAP2 and SNAP25. The predicted FOXC1 binding site is underlined. (B) Agarose gel electrophoresis of HeLa chromatin immunoprecipitated with antibodies for IgG (negative control), H3K4ME3 (active-chromatin marker) and FOXC1. Input sample represents cross-linked but not immunoprecipitated chromatin. C) qPCR results from IgG and FOXC1 immunoprecipitated chromatin for RAB3GAP1, RAB3GAP2, and SNAP25. *P = 0.05.

Fig 2. Luciferase transactivation by FOXC1 through upstream regions of RAB3GAP1, RAB3GAP2 and SNAP25.

Transactivation experiments with plasmid expressing FOXC1 (WT) or mutant FOXC1(p.S131L), and reporter construct (pGL3 or pGL3.TK) containing (A) The 225bp RAB3GAP1 upstream (pGL3.R3G1) and pGL3.FOXO1 (positive control) (B) Upstream region of RAB3GAP1 (pGL3.R3G1), and constructs with each possible combination of putative FOXC1 binding sites deleted. (C) The 211bp RAB3GAP2 upstream region, the region with the BS deleted, pGL3.TK.R3G2.del and pGL3.TK.6xFBS (positive control) (D) The 151bp SNAP25 upstream region (pGL3.TK. SNAP25), and the region with the BS deleted (SNAP25.del). All Experiments were repeated at least three times in triplicate. Error bars represent standard error. N.S Not significant, *P˂0.05, **P˂0.01 versus pGL3.TK or pGL3.

Fig 3. FOXC1 knockdown decreases RNA levels of RAB3GAP1, RAB3GAP2 and SNAP25.

qRT-PCR experiments using RNA isolated from HeLa cells transfected with scrambled siRNA. qPCR was used to evaluate changes in RNA levels of A)FOXC1 B)RAB3GAP1 C)RAB3GAP2, D) Total SNAP25 (SNAP25ab) E) SNAP25 isoform a (SNAP25a), F) SNAP25 isoform b (SNAP25b) and HPRT1 (housekeeping gene control). Fold change in RNA levels was calculated using the ΔΔCt method normalized to HPRT1 and scaled to siRNA scrambled control. Experiments were performed three times in triplicate. Error bars represent standard error. *P˂0.05.

Fig 4. FOXC1 knockdown decreases protein levels of RAB3GAP1, RAB3GAP2 and SNAP25 in HeLa cells.

A) Western blot analysis of HeLa protein lysates after siRNA transfection with siRNA Scrambled control, or siRNA FOXC1. As FOXC1 has a number of phosphorylation sites, a number of bands are observed at ~70kDa, indicated by a curly brace. Blots were probed for FOXC1, RAB3GAP1, RAB3GAP2, SNAP25 and α-Tubulin (loading control). Normalized and scaled values for B) RAB3GAP1 C) RAB3GAP2 and D) SNAP25. All Western blots were repeated at least three times. *P˂0.05, **P˂0.01, ***P<0.001.

Fig 5. FOXC1 over-expression changes protein levels of RAB3GAP1, RAN3GAP2 and SNAP25 in HeLa cells.

Western blot analysis of HeLa protein lysates after transfection with either pcDNA4-Xpress-Empty or pcDNA4-Xpress-FOXC1(WT). Blots were probed with antibodies for FOXC1, RAB3GAP1, RAB3GAP2, SNAP25 and α-Tubulin (loading control). Normalized scaled values were calculated for A) RAB3GAP1 (example blot shown in B). Normalized and scaled values compared to level of FOXC1 knockdown are presented in C) RAB3GAP2 (example blot shown in D) and E) SNAP25 (example blot shown in F) *P˂0.05. Experiments were repeated at least three times.

Fig 6. FOXC1 knockdown decreases intracellular and extracellular levels of exogenous MYOC in HeLa cells.

Western blot analysis using HeLa cell lysate, and cell media collected after transfection with pRc-MYOC (WT) and either siRNA Control or siRNA FOXC1. Blots of lysates were probed and bands were quantified, normalized, and scaled for A) FOXC1, and MYOC using TFIID as a loading control and blots of media were probed for MYOC using ponceau stain intensity as a loading control (right). B) Example blots of lysates (left) and media (right). Error bars represent standard error. *P˂0.05.

Fig 7. RAB3GAP1 knockdown decreases intracellular and increases extracellular levels of exogenous MYOC in HeLa cells.

Western blot analysis using HeLa cell lysate, and cell media collected after transfection with pRc-MYOC (WT) and either siRNA Control or siRNA RAB3GAP1. Blots of lysates were probed and bands were quantified, normalized, and scaled for A) RAB3GAP1, and MYOC using TFIID as a loading control and blots of media were probed for MYOC using ponceau stain intensity as a loading control (right). B) Example blots of lysates (left) and media (right). Error bars represent standard error. *P˂0.05.

Fig 8. RAB3GAP2 knockdown decreases intracellular and extracellular levels of exogenous MYOC in HeLa cells.

Western blot analysis using HeLa cell lysate, and cell media collected after transfection with pRc-MYOC (WT) and either siRNA Control or siRNA RAB3GAP1. Blots of lysates were probed and bands were quantified, normalized, and scaled for A) RAB3GAP2, and MYOC using TFIID as a loading control and blots of media were probed for MYOC using ponceau stain intensity as a loading control (right). B) Example blots of lysates (left) and media (right). Error bars represent standard error. *P˂0.05.

Fig 9. SNAP25 knockdown decreases intracellular and increases extracellular levels of exogenous MYOC in HeLa cells.

Western blot analysis using HeLa cell lysate, and cell media collected after transfection with pRc-MYOC (WT) and either siRNA Control or siRNA RAB3GAP1. Blots of lysates were probed and bands were quantified, normalized, and scaled for A) SNAP25, and MYOC using TFIID as a loading control and blots of media were probed for MYOC using ponceau stain intensity as a loading control (right). B) Example blots of lysates (left) and media (right). Error bars represent standard error. *P˂0.05.

Fig 10. Combined RAB3GAP1, RAB3GAP2 and SNAP25 knockdown decreases intracellular and extracellular levels of exogenous MYOC in HeLa cells.

A) Western blot analysis of intracellular (left) and extracellular (right) MYOC upon knockdown of RAB3GAP1 and RAB3GAP2 in HeLa cells transfected with pRc-MYOC(WT) (B) Example Western blot of A. C) Western blot analysis of intracellular (left) and extracellular (right) MYOC following SNAP25, RAB3GAP1 and RAB3GAP2 knockdown in HeLa cells transfected with pRc-MYOC(WT). D) Example Western blot of C.

Fig 11. Summary of the regulation of RAB3GAP1, RAB3GAP2 and SNAP25 by FOXC1 and its effect on Myocilin secretion.

(A) FOXC1 regulates RAB3GAP1 and RAB3GAP2 which compose the RAB3GAP complex involved in inactivation of RAB3, responsible for bringing secretory vesicles near the plasma membrane. FOXC1 also regulates SNAP25, a member of the SNARE complex, which during exocytosis forms a complex with another v-SNARE and t-SNARE thereby fusing the secretory vesicle with the plasma membrane. (B) FOXC1 knockdown leads to decreased protein levels of RAB3GAP1, RAB3GAP2 and SNAP25 which in turn results in abnormal Myocilin secretion.