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. 2026 May 27;16:24118. doi: 10.1038/s41598-026-54944-9

Persistent loss of membrane-associated MUC4 reprograms physiological functions in corneal epithelial cells via MAPK-dependent cascade

Tsan-Chi Chen 1,2, Wan-Lin Wu 1, Shu-Wen Chang 1,3,4,✉
PMCID: PMC13439477  PMID: 42192195

Abstract

Mucin-4 (MUC4), a key membrane-associated mucin of the corneal apical glycocalyx, plays an important role in maintaining corneal epithelial homeostasis and wettability. Dysregulation of MUC4 has been associated with barrier dysfunction and ocular surface inflammation. This study aimed to investigate the physiological regulatory role of MUC4 in human corneal epithelial cells (HCEs) and elucidate its underlying intracellular mechanisms. MUC4 in HCEs was silenced through lentivirus-based shMUC4 pseudovirion transduction. Cell viability was analyzed via CCK-8 reagent. Cell motility was monitored via real-time culture monitoring system. Mitochondrial function was analyzed by Seahorse analyzer. Expressions of associated proteins were determined by immunoblotting. Changes in phosphorylation levels in major signaling pathways were determined by protein arrays. MUC4 increased time-dependently in HCEs. The expression was efficiently silenced by shMUC4 transduction. MUC4 loss suppressed cell viability through increasing caspase-8 and PARP1 as well as decreasing Bcl-2 and Bcl-xL. MUC4 loss promoted cell motility by increasing the phosphorylation of FAK-Y397 and paxillin-Y118. Maximal respiration and spare respiratory capacity were lower in MUC4-silenced HCEs by decreasing expression of key mitochondrial proteins (ATP8, MT-ND1, MT-ND3, MT-ND5, MT-CO2, and MT-CO3). MUC4 loss activated FAK and triggered the activation of MAPK-associated kinases in HCEs. These findings indicated that persistent MUC4 production maintained cell viability and hindered migration in corneal epithelial cells via FAK/MAPK/P38 signaling pathway.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-54944-9.

Keywords: MUC4, Human corneal epithelial cells, Viability, Motility, Mitochondrial function, MAPK signaling

Subject terms: Cell biology, Diseases, Molecular biology

Introduction

Membrane-associated mucins (MAMs), including mucin-1 (MUC1), mucin-4 (MUC4), and mucin-16 (MUC16), form the apical glycocalyx of the corneal epithelium and create a hydrophilic interface that stabilizes the tear film, reduces friction during blinking, and maintains ocular surface wettability1–4. Beyond their structural functions, MAMs act as sensor–receptors that transduce signals regulating inflammation, cell proliferation, differentiation, apoptosis, and wound healing5–8. Dysregulation of MAMs compromises barrier integrity and promotes chronic inflammation, underscoring their dual protective and pathogenic roles at the ocular surface.

Among MAMs, MUC4 plays a central role in corneal epithelial homeostasis by supporting tear film stability, lubrication, and epithelial barrier function—processes essential in dry eye disease1,9–11. MUC4 is unique among major MAMs in that it does not have a SEA (sea urchin sperm protein, enterokinase, and agrin) domain5,12, resulting in stable anchoring at the epithelial surface. This stability highlights the importance of understanding MUC4-mediated regulation of cell viability, immune responses, and intracellular signaling.

In dry eye disease, quantitative or qualitative defects in MAMs, including MUC4, impair tear film stability and increase susceptibility to epithelial damage1,10,11. MUC4 expression is regulated by environmental and chemical stimuli, and can be upregulated via transient receptor potential vanilloid 4 (TRPV4) activation, a proposed therapeutic target for dry eye13. MUC4 has been shown to be a modulator in regulating cell survival, proliferation, and inflammatory responses in cancer cells14–17. Reduced expression or altered glycosylation of MUC4 contributes to epithelial stress and tear hyperosmolarity, further exacerbating disease pathophysiology1,10,11.

MUC4 is also associated with differentiation and proliferation in corneal epithelial cells18,19. Loss or dysregulation of MUC4 disrupts epithelial barrier integrity and heightens vulnerability to ocular surface stress, key contributors to dry eye pathogenesis9,20. Animal models demonstrate that transcorneal electrical stimulation21 and particulate matter exposure22 disrupt MUC4 layers and lead to dry eye–like changes. Experimental MUC4 knockout results in increased rose bengal staining, surface irregularity, and tear film instability, indicating compromised barrier function9. Upregulation of MUC4 in specific conjunctival regions of dry eye patients suggests a compensatory response to maintain ocular surface homeostasis23. Whether MUC4 contribute to corneal epithelial cell migration remains less well illustrated.

In ocular physiology, the mitogen-activated protein kinase (MAPK) signaling pathway functions as a key transductor for extracellular signals into intracellular responses24. Widely distributed in eukaryotes and highly conserved throughout evolution, MAPK regulates fundamental cellular processes through downstream ribosomal S6 kinase (RSK), mitotic- and stress-activated kinase (MSK)25. Modulation of MAPK signaling mediates crosstalk between P38 and ERK, thereby coordinating cell migration and proliferation during corneal wound repair26. Phosphorylated HSP27 is also a crucial regulator of corneal epithelial cell apoptosis and wound healing27,28. The dynamic coordination of MAPK and P53 controls the balance between cell proliferation and cell death29.

Building on these findings, this study investigates the role of MUC4 in corneal epithelial physiology and evaluates its potential contributions to ocular surface homeostasis.

Results

Culture-associated MUC4 upregulation in HCEs occurs independently of lentiviral transduction

We observed a significant time-dependent increase in MUC4 expression in nontransduced corneal epithelial cells by immunoblotting (Fig. 1). To ensure that the lentiviral transduction process did not interfere with this trend, human corneal epithelial cells (HCEs) were transduced with EGFP (enhanced green fluorescent protein)-expressing lentivirus as a mock group. Comparisons between mock-transduced and nontransduced cells at corresponding time points confirmed that lentiviral-mediated EGFP expression had no adverse effect on MUC4 levels (Fig. 1A and B). These results establish a reliable baseline for subsequent knockdown experiments, confirming that the delivery system itself does not alter the temporal expression patterns of MUC4.

Fig. 1.

Fig. 1

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MUC4 expression increases with incubation time in HCEs. MUC4 expression in (A) nontransduced HCEs (without lentiviral infection) and (C) MUC4-silenced HCEs (shMUC4) was compared with that in mock (EGFP-expressing control for transduction efficiency) HCEs at corresponding time points. (B, D) MUC4 expression levels shown in (A) and (C) were individually normalized to the maximal expression observed in mock HCEs at day 14, which was defined as 100%. Data are presented as mean ± S.D. (n = 3). One-way ANOVA (p values shown in item labels) demonstrated a time-dependent increase in MUC4 expression in mock, nontransduced, and shMUC4 cells, followed by Tukey’s HSD post hoc test (# p < 0.05; ### p < 0.001). Student’s t-test revealed significant differences among shMUC4 cells (** p < 0.01; *** p < 0.001). Based on the prestained ladder, the membranes of each target protein were cut after blocking in 5% BSA/TBST solution. The size of each target protein was indicated in parentheses.

Lentiviral shRNA achieves efficient and stable MUC4 silencing in HCEs

In parallel with the observed temporal increase of MUC4 in HCEs, the efficiency of small hairpin MUC4 (shMUC4)-mediated knockdown was assessed at the same intervals (Fig. 1C and D). MUC4 expression was progressively suppressed, with residual levels of 31.4% on day 7, 27.0% on day 10, and 26.7% on day 14, respectively. These findings demonstrate that MUC4 can be efficiently and stably silenced in HCEs, establishing a reliable MUC4-knockdown cell model for subsequent functional assays.

MUC4 silencing suppresses HCE proliferation without inducing apoptosis

Given the importance of cell proliferation, HCE growth was evaluated using a cell proliferation assay and apoptotic staining (Fig. 2). MUC4 silencing significantly suppressed proliferation over 7-day period post-passaging (p < 0.001), suggesting that loss of MUC4 impairs corneal epithelial cell proliferation (Fig. 2A). Notably, while the positive control (H2O2-treated EGFP-expressed HCEs) exhibited significant apoptosis significant apoptosis, only minimal and negligible apoptotic signaling was observed in either mock or shMUC4 groups, indicating that the observed growth inhibition was not primarily driven by programmed cell death.

Fig. 2.

Fig. 2

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MUC4 loss suppresses cell proliferation in HCEs. (A) Cell viability of mock and MUC4-silenced (shMUC4) HCEs was assessed using the CCK-8 assay. Data was normalized to the mock HCEs on day 1 (set as 1) and presented as mean ± S.D. (n = 3). Statistical significance across time points was determined by one-way ANOVA followed by Tukey’s HSD post hoc test (# p < 0.05; ### p < 0.001). Comparisons between mock and shMUC4 groups at each time point were analyzed using Student’s t-test (** p < 0.01; *** p < 0.001). (B) Representative fluorescence and bright field (BF) images of mock, shMUC4, and positive control HCEs at day 7. The positive control was established by treating EGFP-expressing HCEs with 3% H2O2 for 1 day. TUNEL-positive apoptotic cells are visualized in red, EGFP-overexpressing cells in green, and nuclei are counterstained with DAPI (blue). Images were acquired using a Zeiss fluorescence microscope with specific filter sets (DAPI: 445/50 nm; EGFP: 525/50 nm; TUNEL: 605/70 nm). Scale bar = 100 μm.

To elucidate the molecular mechanisms by which MUC4 maintains cellular homeostasis, we systematically characterized the expression of key survival and apoptosis-related proteins via immunoblotting (Fig. 3A). MUC4 knockdown triggered a distinct shift in the apoptotic machinery, beginning with a modest elevation of caspase-1 (Fig. 3B). Notably, the expression of caspase-8, a pivotal initiator of the extrinsic apoptotic pathway, significantly increased in a time-dependent manner (Fig. 3C), alongside a marked accumulation of full-length PARP1 (Fig. 3D), indicating a heightened cellular stress response. Paradoxically, the levels of cleaved caspase-3 showed a significant decline (Fig. 3E), suggesting that MUC4 deficiency might drive cell death through non-canonical pathways or that executioner caspase activation is transient. This pro-apoptotic shift was further compounded by the collapse of mitochondrial defense, evidenced by the significant downregulation of anti-apoptotic B-cell lymphoma 2 (Bcl-2) family members, Bcl-2 (Fig. 3F) and Bcl-xL (Fig. 3G). Furthermore, the suppression of Cox-2 (Fig. 3H), a key mediator of epithelial survival and proliferative signaling, underscored a diminished regenerative capacity. Collectively, these findings demonstrate that MUC4 serves as a critical molecular rheostat; its loss destabilizes the homeostatic balance by simultaneously augmenting apoptotic priming and debilitating the anti-apoptotic and survival-promoting framework of HCEs.

Fig. 3.

Fig. 3

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MUC4 modulates survival and apoptosis proteins in HCEs. (A) During the same period, expression of cell survival–related proteins (indicated targets) in mock and shMUC4 HCEs was analyzed by immunoblotting. (B–H) Protein expression levels were individually normalized to the maximal expression observed in mock HCEs at day 14 (set to 100%). Data are presented as mean ± S.D. (n = 3). One-way ANOVA (p values shown in item labels) demonstrated time-dependent changes in target protein expression in mock and shMUC4 cells, followed by Tukey’s HSD post hoc test (# p < 0.05; ## p < 0.01; ### p < 0.001). Student’s t-test revealed significant differences among shMUC4 cells (* p < 0.05; ** p < 0.01; *** p < 0.001). Based on the prestained ladder, the membranes of each target protein were cut after blocking in 5% BSA/TBST solution. The size of each target protein was indicated in parentheses.

MUC4 silencing enhances HCE migration via activation of the FAK/paxillin signaling cascade

To assess the role of MUC4 in cell motility, HCE migration was monitored using the ASTEC real-time culture monitoring system at 4 × magnification (Fig. 4A; Supplementary Movies S1 and S2). Compared with mock group, MUC4-silenced cells exhibited significantly faster wound closure, with wound area closing more rapidly over time (Fig. 4B). Immunoblotting identified two key regulators, FAK and paxillin (Fig. 4C), whose expressions increased over time up to 14 days, respectively (Fig. 4D and E). Furthermore, phosphorylation of FAK at Y397 (FAK-pY397; Fig. 4F) and paxillin at Y118 (paxillin-pY118; Fig. 4G) increased significantly in MUC4-silenced cells in a time-dependent manner. These findings suggest that MUC4 acts as a negative regulator of corneal epithelial cell migration, likely by restraining the activation of the FAK/paxillin signaling cascade.

Fig. 4.

Fig. 4

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MUC4 loss accelerates HCEs motility. (A) Cell motility of mock and MUC4-silenced (shMUC4) HCEs was monitored for up to 72 h using a real-time cell culture monitoring system with a 4 × dry objective. Scale bar = 200 μm. (B) Images were analyzed using inForm Advanced Image Analysis software. Curves represent the wound-healing area relative to the full image; quantitative data are presented as mean ± S.D. (n = 5). (C) During the same period, expression and phosphorylation of focal adhesion kinase (FAK) and paxillin in mock and shMUC4 HCEs were analyzed by immunoblotting. Expression of (D) FAK, (E) paxillin, (F) FAK-pY397, and (G) paxillin-pY118 were individually normalized to the maximal value observed in mock HCEs at day 14 (set to 100%). Data are presented as mean ± S.D. (n = 3). One-way ANOVA (p values shown in item labels) demonstrated time-dependent changes in cell motility and target protein expression in mock and shMUC4 cells, followed by Tukey’s HSD post hoc test (# p < 0.05; ## p < 0.01; ### p < 0.001). Student’s t-test revealed significant differences among shMUC4 cells (* p < 0.05; ** p < 0.01; *** p < 0.001). Based on the prestained ladder, the membranes of each target protein were cut after blocking in 5% BSA/TBST solution. The size of each target protein was indicated in parentheses.

MUC4 loss disrupts mitochondrial protein homeostasis and bioenergetic stability in HCEs

Given that mitochondria are the primary energy source driving cellular processes, it is worth considering that mitochondria may be a potential target organelle for MUC4 regulation in HCEs. Mitochondrial function was assessed by measuring changes in oxygen consumption rate (OCR) in MUC4-sileced HCEs (Fig. 5A). This result indicated that mitochondrial function was inhibited in MUC4-silenced HCEs after 4 days of normal incubation, based on the capacity of maximal respiration (Fig. 5B) and spare respiratory capacity (Fig. 5C). This revealed that MUC4 loss impaired mitochondrial activity.

Fig. 5.

Fig. 5

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MUC4 loss reduces mitochondrial function in HCEs. (A) Mitochondrial function in mock and MUC4-silenced (shMUC4) HCEs was determined on day 4 using a Seahorse XFp analyzer for OCR. (B) Maximal respiration and (C) spare respiratory capacity in MUC4-silenced HCEs were calculated and compared to those in mock HCEs. Expression data were presented as the mean ± S.D. (n = 3). Student’s t-test revealed significant differences among shMUC4 cells (*** p < 0.001).

To explore underlying mechanisms, expression of key mitochondrial proteins was analyzed by immunoblotting at matched time points (Fig. 6A). MUC4 loss significantly increased ATP6 expression (Fig. 6B) but suppressed expression of ATP8 (Fig. 6C), MT-ND1 (Fig. 6D), MT-ND5 (Fig. 6F), and MT-CO2 (Fig. 6G). In particular, the expression of MT-ND3 (Fig. 6E) and MT-CO3 (Fig. 6H) in mock group was completely suppressed in MUC4-silenced HCEs. Collectively, these findings demonstrate that MUC4 is critical for maintaining mitochondrial protein homeostasis and overall bioenergetic stability in HCEs.

Fig. 6.

Fig. 6

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MUC4 modulates mitochondrial proteins in HCEs. (A) During the same period, the expression of the mitochondrial proteins (shown as the indicated targets) in mock and MUC4-silenced (shMUC4) HCEs was determined by immunoblotting. (B–H) Expression level of each target was normalized individually against the maximum expression value observed in EGFP-expressed HCEs cultured for 14 days, which was set to 100%. Data are presented as mean ± S.D. (n = 3). One-way ANOVA (p values shown in item labels) demonstrated time-dependent changes in target protein expression in mock and shMUC4 cells, followed by Tukey’s HSD post hoc test (# p < 0.05; ## p < 0.01; ### p < 0.001). Student’s t-test revealed significant differences among shMUC4 cells (* p < 0.05; ** p < 0.01; *** p < 0.001). Based on the prestained ladder, the membranes of each target protein were cut after blocking in 5% BSA/TBST solution. The size of each target protein was indicated in parentheses.

MUC4 regulates corneal epithelial survival and migration through modulation of MAPK signaling

Given the increased phosphorylation of migration-related proteins in MUC4-silenced HCEs, five major signaling pathways—MAPK, AKT, JAK/STAT, NFκB, and TGFβ—were analyzed using a protein array (Fig. 7). MUC4 knockdown selectively enhanced phosphorylation within the MAPK pathway (Fig. 7A and B), while phosphorylation of AKT, JAK/STAT, NFκB, and TGFβ remained largely unaffected (Fig. 7C–F). Within the complex MAPK signaling architecture, MUC4 knockdown appeared to preferentially activate the p38/MAPK pathway. This was characterized by the increased phosphorylation of its upstream activators, MKK3 (1.66-fold) and MKK6 (1.40-fold), as well as the p38 molecule itself (p38, 1.12-fold). Furthermore, several downstream targets associated with the p38 stress-response route exhibited significant activation, including MSK2 (1.50-fold), HSP27 (1.24-fold), P53 (1.27-fold), and the transcription factor CREB (1.75-fold). While phosphorylation of MEK1 (1.40-fold) also increased, the overall profile suggests a selective involvement of the p38-mediated signaling cascade in HCEs following MUC4 silencing. In contrast, other signaling pathways showed less prominent changes. These findings indicate that MUC4 primarily regulates corneal epithelial cell functions, particularly survival and migration, through the modulation of the MAPK signaling architecture.

Fig. 7.

Fig. 7

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MUC4 loss activates MAPK signaling pathway in HCEs. Protein phosphorylation levels in mock and MUC4-silenced (shMUC4) HCEs were assessed on day 4 using the Human Phosphorylation Pathway Profiling Array C55, targeting MAPK (A and B), AKT (C), JAK/STAT (D), NFκB (E), and TGFβ (F) signaling pathways. MAPK signaling pathway expression in mock and MUC4-silenced HCEs is shown in (B). The specific targets are listed in the table on the right, with prominently altered proteins highlighted in red. Expression levels were quantified by normalizing each target to the positive control dots (POS) on each membrane. Data are presented as the mean ± S.D. Student’s t-test revealed significant differences among shMUC4 cells (* p < 0.05).

Discussion

Using shRNA silencing, we demonstrate that loss of MUC4 impaired mitochondrial function, reduced cell viability, and increased cell migration in human corneal epithelial cells (Fig. 8A). These in vitro findings underscore the multifaceted regulatory role of membrane-associated MUC4 in corneal epithelial homeostasis. Consistent with these observations, pathway analysis indicated activation of MAPK signaling following MUC4 depletion. Specifically, loss of MUC4 may converge on FAK activation and subsequent phosphorylation of downstream kinases, culminating in activation of terminal kinases, including MSK2, HSPB1 (HSP27), and P53 (Fig. 8B). These findings suggest that sustained MUC4 loss may modulate MAPK signaling pathways involved in multiple corneal epithelial cell functions. MUC4 downregulation also induced P53 expression, consistent with findings in pancreatic cells14. As MUC4 has been identified as a critical regulator of corneal epithelial cellular responses1,4, we confirmed that reduced MUC4 expression closely associates with dry eye disease and ocular surface damage, contributing to tear film instability and epithelial barrier dysfunction20.

Fig. 8.

Fig. 8

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Regulatory role of membrane-associated MUC4 in human corneal epithelial cells. Predicted pathways illustrate how membrane-associated MUC4 regulates cellular functions (A) and activates MAPK-associated kinases (B). Key expression targets are highlighted in yellow.

MUC4 has been implicated as a modulator of signaling pathways involved in cell survival, proliferation, and inflammatory responses15–17. In corneal epithelial cells, MUC4 has also been associated with differentiation and proliferation, with mature MUC4 predominantly localized to differentiated superficial layers and precursor forms distributed throughout the epithelium18,19. Consistent with these previous observations, MUC4 expression in our culture system increased progressively over time (Fig. 1A and B), reflecting ongoing epithelial maturation. Silencing of MUC4 resulted in a time-dependent decrease in cell proliferation and viability, accompanied by reduced expression of the anti-apoptotic proteins Bcl-2 (Fig. 3F) and Bcl-xL (Fig. 3H). Concurrently, levels of caspase-1 (Fig. 3B), caspase-8 (Fig. 3C), and full-length PARP1 (Fig. 3D) were increased, indicating activation of upstream pro-death/stress signaling. However, cleaved PARP1 was only weakly detected in mock cells and was not significantly elevated following MUC4 knockdown (Fig. 3A), suggesting limited engagement of classical downstream apoptotic execution or involvement of cell death pathways not dependent on PARP1 cleavage.

Similarly, levels of cleaved caspase-3 were reduced following MUC4 knockdown (Fig. 3E), despite its established role as an executioner caspase in apoptosis. Several studies have shown that MUC4 is involved in cell survival, proliferation, and inflammatory responses15–17. Accumulating evidence indicates that caspase-3 can also participate in nonapoptotic processes, including regulation of differentiation, proliferation, and cellular stress responses, when its activation is limited or tightly controlled30,31. In addition, caspase-3 activity can promote cell proliferation during the regeneration phase32. Together, these findings raise the possibility that MUC4 loss induces a stress-primed state characterized by activation of upstream death signaling without robust activation of classical execution-phase markers (eg, cleaved caspase-3 and cleaved PARP1). Such dysregulated, sublethal caspase signaling has been reported in epithelial stress responses and may contribute to impaired proliferation, mitochondrial dysfunction, and reduced cell survival. Collectively, our results support a model in which MUC4 promotes corneal epithelial viability at least in part by sustaining anti-apoptotic signaling and limiting initiation of maladaptive stress responses.

Furthermore, Cox-2 has also been implicated in corneal epithelial proliferation, particularly in response to injury or inflammatory stress, through prostaglandin-mediated growth signaling33. In our model, MUC4 silencing markedly reduced Cox-2 expression in HCEs (Fig. 3H). This reduction, together with observed alterations in apoptotic and pro-survival-associated regulators, further supports a role for MUC4 as a stabilizing factor that promotes corneal epithelial cell viability.

Corneal epithelial cell migration is essential for wound repair and is regulated by signaling pathways that also influence mucin expression8. Although membrane-associated mucins primarily contribute to barrier formation and lubrication, their expression in corneal epithelial cells is closely associated with epithelial differentiation, a process modulated by growth factors such as FGF1034. Notably, FGF10 has been shown to enhance mucin expression while simultaneously promoting epithelial migration, highlighting coordinated regulation of these processes. In contrast to reports demonstrating that MUC4 promotes migration and metastasis in multiple epithelial cancers, including pancreatic35, breast36, colon, ovarian, and prostate16, our results indicate an opposing role in corneal epithelial cells. MUC4-silenced HCEs exhibited significantly increased migratory capacity compared with mock group (Fig. 4A and B; Supplementary Movies S1 and S2). The observed enhancement in migration correlated with elevated phosphorylation levels of FAK (Fig. 4C and F) and paxillin (Fig. 4C and G). The FAK/paxillin signaling cascade is a critical pathway governing the migration of various cell types, as evidenced by prior studies and our own findings in corneal cells37–39. This suggests a potential link between MUC4 expression and the modulation of the FAK/paxillin axis, where the attenuation of this signaling pathway during cellular maturation contributes to reduced corneal epithelial cell motility. Our results could be explained by the fact that corneal epithelial migration is highest in immature, proliferative cells and decreases as cells undergo terminal differentiation and maturation40,41. Also, as we showed MUC4 increased along with cellular maturation in culture (Fig. 1).

Reduced expression of mitochondrial proteins like MT-COs and MT-NDs is linked to bioenergetic failure at the ocular surface42. Consistently, MUC4 depletion led to a decline in mitochondrial function (Fig. 5), evidenced by the coordinated downregulation of Complex I (MT-ND1, MT-ND3, MT-ND5), Complex IV (MT-CO2, MT-CO3), and Complex V (ATP8) (Fig. 6). Notably, MT-CO2 is essential for cytochrome c oxidase activity and ATP production; its loss limits the energy available for barrier maintenance and epithelial repair. The transient increase in MT-ATP6, followed by a decline after day 7 (Fig. 6B), may represent a short-lived compensatory response to respiratory chain dysfunction43,44. Such bioenergetic failure severely impairs corneal homeostasis and oxidative stress resistance, leading to the suppression of proliferation observed in our study (Fig. 2). These findings suggest that MUC4 acts as a key regulator of mitochondrial proteostasis, and its deficiency initiates a cascade of metabolic impairments that compromise the overall health of the corneal epithelium, as seen in disorders like keratoconus45,46.

Furthermore, among signaling pathways regulating epithelial survival and stress responses, MUC4 knockdown selectively increased phosphorylation of MAPK pathway components (Fig. 7A and B), whereas phosphorylation of AKT, JAK/STAT, NFκB, and TGFβ signaling proteins was not significantly altered (Fig. 7B–E). Although MUC4 has been implicated in multiple signaling networks, including ERBB, NFκB, Wnt/β-catenin, PI3K/AKT, MAPK/ERK, and JAK/STAT5–7, our results indicate a selective correlation between MUC4 loss and the activation of FAK/paxillin and MAPK/p38 signaling cascades. Given the role of MAPK signaling in epithelial migration, wound repair, and differentiation, enhanced MAPK activation following MUC4 depletion may underlie downstream alterations in barrier-related gene expression, such as keratin 10 (K10) and paired box 6 (Pax6)9,24–26. While these findings point toward a potential regulatory link where metabolic stress might trigger signaling shifts to promote cell motility, further investigation is required to definitively establish whether these phenotypic changes are directly mediated through these specific pathways.

Interestingly, we observed variability in MUC4 expression in HCEs following initial seeding and post-thaw recovery, underscoring that standard culture procedures can influence cellular characteristics over time. However, there is limited comparative evidence addressing MUC4 stability dynamics in HCE models, highlighting the need for more detailed characterization. Notably, despite the potential for lentiviral-mediated off-target effects, our data demonstrate that intracellular MUC4 levels in the EGFP transduced mock group remained comparable to nontransduced cells (Fig. 1). This specificity is reinforced by the stable expression of other common proteins, consistent with our previous work47. In addition, we observed a reproducible increase in intrinsic MUC4 expression over a 7-day period following HCE seeding, consistent with progressive epithelial maturation in culture. This temporal pattern correlates with the maturation timeline of tight junction structures, which typically reach completion between days 4 and 948. Given that these immortalized HCEs are a well-validated platform for corneal physiology49,50, this culture-associated fluctuation in MUC4 expression may partially mirror MUC4 dysregulation reported in ocular surface pathologies. Consequently, this system provides a robust in vitro model for investigating the physiological consequences of MUC4 modulation.

The role of MUC4 in corneal injuries has been highlighted in several studies, where its expression is often modulated in response to epithelial damage51,52. For instance, MUC4 has been shown to be downregulated during the early stages of corneal wound healing, only to be restored as the epithelial barrier reestablishes53. This aligns with our observation that MUC4 silencing accelerates migration, potentially as a compensatory mechanism to cover denuded areas, albeit at the cost of reduced proliferative capacity and bioenergetic stability. Furthermore, in models of dry eye and chemical burns, the loss of MUC4 is closely associated with barrier dysfunction and delayed epithelial recovery9,53–55. Our findings extend these observations by demonstrating that MUC4 maintenance is critical for mitochondrial proteostasis and metabolic homeostasis, providing a new mechanistic perspective on why MUC4 deficiency complicates the recovery process in clinical corneal injuries.

While our findings provide new insights into the role of MUC4 in corneal epithelial physiology and its implications for dry eye pathophysiology, several limitations should be noted. Primarily, the use of an immortalized corneal epithelial cell line may not fully recapitulate the complexity of the ocular surface environment. While this model offers a reproducible system for mechanistic investigation, it cannot entirely mimic tissue homeostasis, tear film interactions, or epithelial turnover. Therefore, future studies utilizing primary human corneal epithelial cells, ex vivo tissues, or in vivo models are essential to validate the clinical relevance of our findings. Additionally, while we observed that MUC4 knockdown enhanced cell motility (migration) and selectively enhanced MAPK activation, whether these changes directly contribute to epithelial barrier disruption or wound healing defects remain not fully elucidated. Further research involving pathway-specific modulation will be necessary to clarify the precise mechanistic contributions of MUC4 to ocular surface homeostasis.

Conclusions

In summary, our findings demonstrate that MUC4 is a critical regulator of corneal epithelial homeostasis. Loss of MUC4 compromises epithelial cell viability and mitochondrial function while promoting aberrant cell migration. These effects are accompanied by induction of incomplete apoptotic stress, suppression of pro-survival signaling, and selective activation of the MAPK pathway. Collectively, these data identify MUC4 as a stabilizing factor that coordinates energy metabolism, survival, and migratory behavior in corneal epithelial cells, and suggest that disruption of MUC4 might contribute to impaired wound healing and ocular surface dysfunction.

Methods

Cell cultivation

HCEs are an immortalized cell line, HCE-2 [50.B1] (catalog number CRL-3582, produced from a descendant of ATCC CRL-11135), originally from the American Type Culture Collection (ATCC, Manassas, VA). Ethical approval was not required for this study. The cells were cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY), 1% nonessential amino acids (Life Technologies, Grand Island, NY), 2 ng/ml epidermal growth factor, 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma‒Aldrich, St. Louis, MO). Cells were maintained at 37 °C in a 5% CO2 environment with the medium refreshed every 2–3 days.

To assess time-dependent effects, cells were seeded at a consistent density of 1 × 106 cells per 6-cm culture dish, and various analyses were performed at specific intervals (Days 1, 4, 7, 10, and 14 post-seeding). To ensure experimental reproducibility and minimize passage-dependent drift, all experiments were conducted using cells within five passages of thawing.

MUC4 silencing

To knock down MUC4 or express enhanced green fluorescent protein (EGFP; serving as a mock control to provide a visual indicator of transduction efficiency while remaining biologically inert regarding MUC4 regulation) in HCEs, lentiviral plasmids containing shMUC4 (TRCN0000123299; 5’-CCTCATCCTTACCGCACATTT-3’) or EGFP (pAS2.EGFP) were obtained from National RNAi Core Facility, Academia Sinica, Taiwan. Pseudovirions were produced in 293 T cells (CRL-11268; ATCC) co-transfected with pCMV△R8.91, pMD.G, and either pLKO.1-shMUC4 or pAS2.EGFP using X-tremeGENE HP DNA Transfection Reagent (Roche Applied Science, Indianapolis, IN). HCEs were infected with shMUC4- or EGFP-containing pseudovirions at a multiplicity of infection of 1. For stable expression, infected cells were maintained under standard culture conditions with 1 μg/mL puromycin (Sigma-Aldrich)47.

Cell viability

Cell proliferation of MUC4-silenced and mock HCEs was assessed using Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) according to manufacturer’s instructions with minor modifications. Briefly, 1 × 104 cells were seeded per well in a 96-well plate (Day 0). After cell attachment on Day 1, fresh medium containing 10 μL CCK-8 reagent was added, and cells were incubated at 37 °C for 2 h. Absorbance was measured at 450 nm with a 690 nm reference using a spectrophotometer.

TUNEL staining

To assess apoptotic signaling, DNA fragmentation was visualized using an In Situ Cell Death Detection Kit, TMR red (Roche Diagnostics GmbH), according to the manufacturer’s protocol with minor modification. Briefly, 2 × 105 cells were seeded in 3-cm culture dishes, respectively. During analysis, cells were washed with phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) and fixed with 4% paraformaldehyde for 1 h at room temperature. Subsequently, cells were washed with PBS and incubated in a fresh permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 15 min on ice. Finally, cells were incubated with the TdT enzyme reaction mixture in a humidified incubator at 37 °C for 60 min in the dark. Apoptotic signals were visualized and captured using a Zeiss fluorescence microscope (Carl Zeiss, Oberkochen, Germany). Images were acquired at an excitation wavelength of 550/25 nm and an emission wavelength of 605/70 nm.

Cell migration assay

Cell migration was evaluated using Oris Cell Migration Assay (Amsbio, Cambridge, MA). HCEs (1 × 104 cells per well) were seeded into 96-well plates with Oris Cell Seeding Stoppers in place and allowed to attach and reach confluence under standard culture conditions by Day 4. Migration of MUC4-silenced and mock cells was monitored and imaged using a real-time culture monitoring system with multiple-point imaging (CCM-MULTI; ASTEC, Fukuoka, Japan). The captured images were analyzed by inForm advanced image analysis software (PerkinElmer Inc., Hopkinton, MA).

Oxygen consumption rate

Oxygen consumption rate (OCR) was measured in HCEs using a Seahorse XFp Analyzer (Agilent Technologies, Santa Clara, CA) according to manufacturer’s instructions with minor modifications. Briefly, 1 × 104 cells were seeded per well in Seahorse XF miniplates and cultured until Day 4. Cells were sequentially treated with oligomycin, FCCP, and rotenone/antimycin A, and real-time changes in OCR were recorded and analyzed using Seahorse Wave software.

Immunoblotting

Protein expression was analyzed by immunoblotting. HCEs were washed with phosphate-buffered saline and lysed in buffer containing 50 mM Tris–HCl, 150 mM NaCl, 0.5% sodium deoxycholate, 1% IGEPAL CA-630, 0.2% SDS, pH 7.4, with protease and phosphatase inhibitors (Roche Diagnostics GmbH). Protein concentrations were determined using BCA assay (Pierce, Rockford, IL), and equal amounts of lysate were mixed with 3 × sample buffer (180 mM Tris–HCl, 30% glycerol, 6% sodium deoxycholate, 3.75% β-mercaptoethanol, pH 6.8). Ten micrograms of each protein sample and two microliters of PageRuler Prestained Protein Ladder (#26,616; Thermo Fisher Scientific, Inc., Waltham, MA) were separated by SDS-PAGE and then transferred to a PVDF membrane using a Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad Laboratories, Inc., Hercules, CA) at a steady current of 2 mA/cm2 gel area for 3 h. According to the prestained ladder, the membrane of each target protein was cut separately. All membranes were then blocked in 5% bovine serum albumin (BSA)/Tris-buffered saline with Tween-20 (TBST; 20 mM Tris–HCl, 137 mM NaCl, 0.05% Tween-20, pH7.6) for 1 h and incubated overnight at 4 °C with primary antibodies against ATP6 (ab190287), ATP8 (ab243667), Bcl-2 (ab32124), Bcl-xL (ab32370), caspase-1 (ab179515), caspase-8 (ab32397), Cox-2 (ab15191), FAK (ab131435), FAK-pY397 (ab81298), MT-CO2 (ab79393), MT-CO3 (ab110259), MT-ND1 (ab181848), MT-ND2 (ab192306), MT-ND5 (ab138136), MUC4 (ab194363), PARP1 (ab32138), cleaved PARP1 (ab32561) (Abcam, Waltham, MA), β-actin (sc-130065), GFP (sc-8334), paxillin (sc-5574), paxillin-pY118 (sc-101774) (Santa Cruz Biotechnology Inc., Dallas, TX), and caspase-3 (#9668) (Cell Signaling Technology Inc., Danvers, MA), with dilutions of 1:1000 in 5% BSA/TBST. After washing three times with TBST for 20 min each time, the membranes were incubated with their specific HRP-conjugated secondary antibodies against goat (ab7125), mouse (ab97040), and rabbit (ab7090) (Abcam), with diluted 1:5000 in 5% BSA/TBST for 1 h at room temperature. Finally, each blot was individually developed using ECL Prime (Amersham, Buckinghamshire, UK) and cumulatively imaged in ten-second increments using a FUJIFILM ImageQuant LAS-4000 system (FujiFilm, Tokyo, Japan). After selecting a suitable image (Supplementary Fig. S1), the densitometry analysis was performed using Multi Gauge V3.0 software (FujiFilm).

Expression of phosphorylated proteins

To assess activation of five key regulatory kinases, phosphorylated protein levels in HCE lysates were measured using Human Phosphorylation Pathway Profiling Array C55 (RayBiotech, Peachtree Corners, GA) following manufacturer’s instructions with minor modifications. The array profiles included MAPK, AKT (protein kinase B), JAK (Janus kinases)/STAT (signal transducer and activator of transcription), NFκB (nuclear factor-kappa B), and TGFβ (transforming Growth Factor-beta) signaling pathways. Dot intensities were imaged with FUJIFILM ImageQuant LAS-4000 system and quantified using Multi Gauge V3.0 software (FujiFilm).

Statistical analysis

All experiments were performed at least triplicate according to experimental requirements. Data were presented as mean ± standard deviation (S.D.). Statistical analyses were conducted using SPSS software (version 26; IBM Corp., Armonk, NY). Differences in specified molecular targets among time points within mock, nontransduced, or MUC4-silenced groups were analyzed using one-way ANOVA followed by Tukey’s honestly significant difference (HSD) post hoc test. Comparisons between two groups under identical conditions at specific time points were performed using Student’s t-test. A p value < 0.05 was considered statistically significant.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (13.8MB, mp4)
Supplementary Material 2 (10.6MB, mp4)
Supplementary Material 3 (111.1KB, pdf)
Supplementary Material 4 (2.2MB, pdf)

Acknowledgements

The authors thank core laboratory of Far Eastern Memorial Hospital, Taiwan and National RNAi Core Facility, Taiwan for technical support.

Author contributions

T.C. and S.C. designed the experiment(s); T.C. conducted the experiment(s); All authors analyzed/interpreted data and wrote/proofed/revised the manuscript.

Funding

This study was supported by Far Eastern Memorial Hospital, Taiwan [FEMH-2021-C-004, FEMH-2022-C-062, FEMH-2024-C-049, FEMH-108-2314-B-418-015, FEMH-109-2314-B-418-005, FEMH-110-2314-B-418-012-MY3, FEMH-113-2314-B-009, FEMH-114-2314-B-014] and National Science and Technology Council, Taiwan [MOST-108-2314-B-418-015, MOST-109-2314-B-418-005, MOST-110-2314-B-418-012-MY3, NSTC-113-2314-B-009, NSTC-114-2314-B-014].

Data availability

Data is available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare that they have no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

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Supplementary Materials

Supplementary Material 1 (13.8MB, mp4)
Supplementary Material 2 (10.6MB, mp4)
Supplementary Material 3 (111.1KB, pdf)
Supplementary Material 4 (2.2MB, pdf)

Data Availability Statement

Data is available from the corresponding author on reasonable request.

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