The conjunctiva is a mucous membrane that covers the surfaces of the eyes and eyelids. The conjunctival epithelial cell layer forms a non-keratinized, stratified columnar epithelium with characteristics different from the eyelid epidermis and cornea. The conjunctival epithelium differs from the cornea in that it comprises two types of cells, epithelial and goblet cells, which are heterogeneously distributed. The cornea and conjunctiva can be discriminated by their expression of different subsets of cytokeratins, KRT12 and KRT13, respectively. The conjunctival epithelium is also instrumental in supporting the tear film by membrane-associated mucins on its cell surface (MUC1, MUC4, MUC16) and by mucins secreted by goblet cells (MUC5AC), maintaining the homeostasis of the conjunctival epithelium (Freddo and Chaum, 2018).

During development, a distinguishing feature of the ocular versus epidermal epithelia is PAX6, a transcription factor expressed throughout the conjunctival and corneal epithelium until adulthood (Koroma et al., 1997). PAX6 is a transcription factor with two independent DNA-binding domains: the paired domain (PD) and paired-type homeodomain (Chi and Epstein, 2002). The PD is divided into an N-terminal PAI (PAIred) and a C-terminal RED (paiRED) domain. When exon 5a is alternatively spliced, it inserts a sequence of 14 amino acids into the PAI domain of PAX6 isoform a (PAX6a), resulting in PAX6 isoform b (PAX6b). Therefore, PAX6a and PAX6b may exert different transcriptional activities, as demonstrated for corneal genes (Sasamoto et al., 2016), but their relative abundance and functional differences in the conjunctiva are unclear. Clinically, loss of PAX6 function leads to conditions such as aniridia and limbal stem cell deficiency (Latta et al., 2021), but the molecular roles of this transcription factor in eye physiology remain enigmatic.

Dry eye syndrome (DES) is a multifactorial clinical complication that affects conjunctival homeostasis (Milner et al., 2017). A major cause of DES is tear film production and maintenance machinery dysfunction, mainly through chronic inflammation, as seen in severe ocular surface diseases, such as Sjögren's syndrome and Stevens-Johnson syndrome (Ogawa, 2018). This may cause keratinized squamous metaplasia, a pathological transition from ocular to epidermal epithelia (Li et al., 2008a, Li et al., 2008b). Interestingly, squamous metaplasia of the ocular surface epithelium is also accompanied by a loss of PAX6 expression (Chen et al., 2013), but its causal connections remain unclear.

Given the scarcity of a cell line model that would enable us to specifically interrogate the ocular surface epithelium in detail, we previously established a conditionally immortalized conjunctival epithelial cell model, iHCjEC (Mitani et al., 2019). In the presence of doxycycline (DOX), iHCjEC proliferation is enhanced by the overexpression of SV40 large T antigen and telomerase reverse transcriptase (TERT). The omission of DOX reduces the expression of large T antigen and restores quasi-normal cell-cycle progression. iHCjECs can stratify when provided an underlying rabbit stromal feeder and insert-cultured in a culture media supplemented with serum. Preliminary molecular profiling of iHCjECs revealed the expression of conjunctival cytokeratins and membrane-associated mucins, but no corneal markers, suggesting the retention of conjunctival traits versus those of other surface epithelia. However, some components of the normal conjunctiva, such as goblet cells, were not completely replicated (Mitani et al., 2019). This validates iHCjECs as a conjunctival epithelial cell model, but the exact events leading to the complete reinstruction of the conjunctival traits remains elusive.

In this study, we conducted systematic transcriptomic analyses to better understand the nature of iHCjECs and to obtain clues for iHCjEC reinstruction to restore several conjunctival characteristics. We reveal that immortalization-cessation, with attenuated SV40 large T antigen function, derepressed p53-activity, inducing an expression profile linked to cell senescence. Somewhat unexpectedly, immortalization-cessation also exhibited a strong tendency of keratinization/cornification, a process unique to the skin epidermis. The sustained expression of conjunctival/epidermal stem cell-associated markers in iHCjECs, along with the lack of corneal markers, hint at a conjunctival–epidermal transition.

Because iHCjECs are cultured on a collagen-coated surface devoid of mesenchymal cells, the loss of mesenchymal–epithelial interactions may contribute to epidermal metaplasia or the loss of PAX6 expression (Kobayashi et al., 2015; Pearton et al., 2005). Previous studies have suggested that epidermal growth factor (EGF) and fibroblast growth factor 7 (FGF7, also known as keratinocyte growth factor) determine conjunctival cell fate during human pluripotent stem-cell differentiation (Hayashi et al., 2016; Nomi et al., 2021), and that human corneal fibroblasts secrete FGF7 and EGF (Kobayashi et al., 2015). Therefore, we investigated the effects of EGF and FGF7 on iHCjECs, as well as the effects of directly introducing PAX6 to iHCjECs.

In clinics, steroids attenuate the underlying inflammatory responses leading to DES and severe ocular surface diseases. We chose dexamethasone (DEX), a synthetic glucocorticoid used to manage systemic inflammation and the ensuing keratinized squamous metaplasia (Coursey and de Paiva, 2014; Matsumoto et al., 2023; Periman et al., 2020; Pflugfelder, 2004), and observed its effects on iHCjECs. This allowed the assessment of DEX administered directly to iHCjECs without the presence of any immune cells, as few studies have directly evaluated the effects of DEX on ocular surface epithelia.

Autologous serum eye drops are used for dry eyes and inflammatory conditions owing to their biochemical similarities to tears (Cui et al., 2021; Quan et al., 2023). However, few studies have considered the possibility that the components in serum might have instructive effects on ocular surface epithelia. Therefore, we also evaluated the direct effects of serum on iHCjECs in an artificial environment devoid of immune cells.

We utilized this spontaneous keratinized metaplasia model of conjunctival epithelia to molecularly dissect the effects of candidate mesenchymal factors (Kobayashi et al., 2015), PAX6 administration (Sun et al., 2018), and clinically used interventions (steroids and serum) (Quan et al., 2023) on epithelia.