TGFβ signaling inhibits goblet cell differentiation via SPDEF in conjunctival epithelium

Abstract

The ocular surface epithelia, including the stratified but nonkeratinized corneal, limbal and conjunctival epithelium, in concert with the epidermal keratinized eyelid epithelium, function together to maintain eye health and vision. Abnormalities in cellular proliferation or differentiation in any of these surface epithelia are central in the pathogenesis of many ocular surface disorders. Goblet cells are important secretory cell components of various epithelia, including the conjunctiva; however, mechanisms that regulate goblet cell differentiation in the conjunctiva are not well understood. Herein, we report that conditional deletion of transforming growth factor β receptor II (Tgfbr2) in keratin 14-positive stratified epithelia causes ocular surface epithelial hyperplasia and conjunctival goblet cell expansion that invaginates into the subconjunctival stroma in the mouse eye. We found that, in the absence of an external phenotype, the ocular surface epithelium develops properly, but young mice displayed conjunctival goblet cell expansion, demonstrating that TGFβ signaling is required for normal restriction of goblet cells within the conjunctiva. We observed increased expression of SAM-pointed domain containing ETS transcription factor (SPDEF) in stratified conjunctival epithelial cells in Tgfbr2 cKO mice, suggesting that TGFβ restricted goblet cell differentiation directly by repressing Spdef transcription. Gain of function of Spdef in keratin 14-positive epithelia resulted in the ectopic formation of goblet cells in the eyelid and peripheral cornea in adult mice. We found that Smad3 bound two distinct sites on the Spdef promoter and that treatment of keratin 14-positive cells with TGFβ inhibited SPDEF activation, thereby identifying a novel mechanistic role for TGFβ in regulating goblet cell differentiation.

Three cheers for the goblet cell: maintaining homeostasis in mucosal epithelia

“…Klf4 and Klf5 mRNA levels were unchanged in microarray analysis of the conjunctiva of Spdef null mice [23], indicating that Klf4 and Klf5 function upstream of Spdef ( Figure 4A), while microarray analysis of the conjunctiva of Klf4 deficient mice showed a decrease in Spdef expression as well as of the winged-helix transcription factors Foxa1 (forkhead box A1) and Foxa3 [5]. Foxa3 and SPDEF mutually induce each other's expression in keratin 14-expressing cells in vitro [24].…”

“…One gene that is of special interest in goblet cell differentiation is sterile a motif pointed domain epithelial specific transcription factor (Spdef). SPDEF was originally described as a prostate-specific transcription factor [18,19], but has since been shown to play an integral role in goblet cell differentiation in the intestine [20,21], the lung [22], and the conjunctiva [23,24] (Box 1). However, upstream regulators of Spdef vary contextually or have not been tested globally, thus hindering a complete understanding of goblet cell differentiation.…”

“…Loss of Spdef results in impairment of goblet cell maturation in the intestine [20], failure of goblet cell induction in response to allergen in the lung [82], and complete loss of goblet cell differentiation in the conjunctiva [23]. Conversely, expression of SPDEF results in an expansion of goblet cells at the expense of other cell types in the intestine [21], the lung [22], and the conjunctiva [24]. It is clear that SPDEF plays an integral role in the differentiation and maturation of goblet cells; however, upstream regulators of Spdef vary substantially in a tissue-dependent manner.…”

“…In the lung, activated b-catenin inhibits the expression of Foxa2, promoting goblet cell metaplasia [80] ( Figure 3A). Murine conjunctival goblet cells express the secreted Wnt inhibitor Frzb (frizzledrelated protein), suggesting that the Wnt signaling [24]. Abbreviation: f, fornix.…”

Defective eyelid leading edge cell migration in C57BL/6-corneal opacity mice with an “eye open at birth” phenotype

“…After E16.5, eyelid growth was slowed during development in B6-Co mice, and the eyelids remained open even at P0 (Figure 1E-H). It has been reported that the best time for eyelid closure is about E16.5, at which time the cornea and other eye accessory structures involved in gradual development could interfere with fusion of the upper and lower eyelids (McCauley et al, 2014;Heude et al, 2015). The eyelids of B6-Co mice grew slowly, and therefore missed the ideal period for eyelid fusion, demonstrating temporal and spatial specificity in embryonic development.…”