RHBDF2-Regulated Growth Factor Signaling in a Rare Human Disease, Tylosis With Esophageal Cancer: What Can We Learn From Murine Models?

Tylosis with esophageal cancer syndrome (TOC) is a rare autosomal dominant proliferative skin disease caused by missense mutations in the rhomboid 5 homolog 2 (RHBDF2) gene. TOC is characterized by thickening of the skin in the palms and feet and is strongly linked with the development of esophageal squamous cell carcinoma. Murine models of human diseases have been valuable tools for investigating the underlying genetic and molecular mechanisms of a broad range of diseases. Although current mouse models do not fully recapitulate all aspects of human TOC, and the molecular mechanisms underlying TOC are still emerging, the available mouse models exhibit several key aspects of the disease, including a proliferative skin phenotype, a rapid wound healing phenotype, susceptibility to epithelial cancer, and aberrant epidermal growth factor receptor (EGFR) signaling. Furthermore, we and other investigators have used these models to generate new insights into the causes and progression of TOC, including findings suggesting a tissue-specific role of the RHBDF2-EGFR pathway, rather than a role of the immune system, in mediating TOC; and indicating that amphiregulin, an EGFR ligand, is a functional driver of the disease. This review highlights the mouse models of TOC available to researchers for use in investigating the disease mechanisms and possible therapies, and the significance of genetic modifiers of the disease identified in these models in delineating the underlying molecular mechanisms.

ADAM17 is essential for ectodomain shedding of the EGF-receptor ligand amphiregulin.

The epidermal growth factor (EGF)-receptor ligand amphiregulin (AREG) is a potent growth factor implicated in proliferative skin diseases and in primary and metastatic epithelial cancers. AREG, synthesized as a propeptide, requires conversion to an active peptide by metalloproteases by a process known as ectodomain shedding. Although (ADAM17) a disintegrin and metalloprotease 17 is a key sheddase of AREG, ADAM8-, ADAM15-, and batimastat (broad metalloprotease inhibitor)-sensitive metalloproteases have also been implicated in AREG shedding. In the present study, using a curly bare (Rhbdf2cub ) mouse model that shows loss-of-hair, enlarged sebaceous gland, and rapid cutaneous wound-healing phenotypes mediated by enhanced Areg mRNA and protein levels, we sought to identify the principal ectodomain sheddase of AREG. To this end, we generated Rhbdf2cub mice lacking ADAM17 specifically in the skin and examined the above phenotypes of Rhbdf2cub mice. We find that ADAM17 deficiency in the skin of Rhbdf2cub mice restores a full hair coat, prevents sebaceous gland enlargement, and impairs the rapid wound-healing phenotype observed in Rhbdf2cub mice. Furthermore, in vitro, stimulated shedding of AREG is abolished in Rhbdf2cub mouse embryonic keratinocytes lacking ADAM17. Thus, our data support previous findings demonstrating that ADAM17 is the major ectodomain sheddase of AREG.

Early induction of NRF2 antioxidant pathway by RHBDF2 mediates rapid cutaneous wound healing.

Rhomboid family protein RHBDF2, an upstream regulator of the epidermal growth factor (EGF) receptor signaling, has been implicated in cutaneous wound healing. However, the underlying molecular mechanisms are still emerging. In humans, a gain-of-function mutation in the RHBDF2 gene accelerates cutaneous wound healing in an EGFR-dependent manner. Likewise, a gain-of-function mutation in the mouse Rhbdf2 gene (Rhbdf2cub/cub) shows a regenerative phenotype (rapid ear-hole closure) resulting from constitutive activation of the EGFR pathway. Because the RHBDF2-regulated EGFR pathway is relevant to cutaneous wound healing in humans, we used Rhbdf2cub/cub mice to investigate the biological networks and pathways leading to accelerated ear-hole closure, with the goal of identifying therapeutic targets potentially effective in promoting wound healing in humans. Comparative transcriptome analysis of ear pinna tissue from Rhbdf2cub/cub and Rhbdf2+/+ mice at 0h, 15min, 2h, and 24h post-wounding revealed an early induction of the nuclear factor E2-related factor 2 (NRF2)-mediated anti-oxidative pathway (0h and 15min), followed by the integrin-receptor aggregation pathway (2h) as early-stage events immediately and shortly after wounding in Rhbdf2cub/cub mice. Additionally, we observed genes enriched for the Fc fragment of the IgG receptor IIIa (FCGR3A)-mediated phagocytosis pathway 24h post-wounding. Although cutaneous wound repair in healthy individuals is generally non-problematic, it can be severely impaired due to aging, diabetes, and chronic inflammation. This study suggests that activation of the NRF2-antioxidant pathway by rhomboid protein RHBDF2 might be beneficial in treating chronic non-healing wounds.

The effect of paraformaldehyde fixation on the delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) measurement.

The delayed Gadolinium-Enhanced Magnetic Resonance Imaging of Cartilage (dGEMRIC) method allows for both qualitative and quantitative measurement of the spatial distribution of glycosaminoglycan [GAG] in excised cartilage. The objective of this study was to determine the effect of paraformaldehyde fixation on dGEMRIC measurements. Five bovine and seven human cartilage pieces were punched into 5-mm plugs, fixed for 18 h in 4% paraformaldehyde solution, and washed. The magnetic resonance imaging (MRI) parameter T1 was measured prior and post fixation in cartilage without (T1(0)) and with (T1(Gd)), the ionically charged MRI contrast agent Gd(DTPA)(2-). Images of tissue before and after fixation were qualitatively very similar. The ratios of T1(0), T1(Gd), and calculated [GAG] after fixation, relative to before fixation, were near or slightly higher than 1 for both bovine cartilage (1.01 +/- 0.01, 1.04 +/- 0.02, 1.05 +/- 0.03, respectively) and for human cartilage (0.96 +/- 0.11, 1.03 +/- 0.05, 1.09 +/- 0.13). Thus, these data suggest that dGEMRIC can be used on previously fixed samples to assess the three dimensional spatial distribution of GAG.

Relationship between cartilage stiffness and dGEMRIC index: correlation and prediction.

We sought to determine if a generalized relationship between the dGEMRIC index (T1Gd relaxation time) and compressive stiffness of articular cartilage could be defined across multiple samples. Osteochondral blocks were cut from 12 human tibial plateaus, six from cadaveric sources and six from total knee replacement surgeries. Each block contained submeniscal ("covered") and extrameniscal ("uncovered") cartilage regions. At approximately 18 sites per block, a pulse indentation was applied and local dGEMRIC index was determined using dGEMRIC MRI. No correlation was found between stiffness and full cartilage depth dGEMRIC index. When averaged over the depth comparable to the indentation, good correlations were found between stiffness and the dGEMRIC index whether all data were combined, or whether each sample/region was considered independently. However, the standard error of the estimate for predicting stiffness from the local dGEMRIC index was improved when the uncovered and covered regions were considered separately. Further improvement in predictive capacity was obtained if, rather than predict absolute stiffness, differences in the dGEMRIC index across a region were used to predict difference in stiffness. The dGEMRIC index is highly correlated to mechanical stiffness. A generalized relationship was found to provide good correspondence across sources and regions. Use of the dGEMRIC index as a predictive measure of stiffness is possible, depending on the application's acceptable error.