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.

A cationic gadolinium contrast agent for magnetic resonance imaging of cartilage.

A new cationic gadolinium contrast agent is reported for delayed gadolinium enhanced magnetic resonance imaging of cartilage (dGEMRIC). The agent partitions into the glycosaminoglycan rich matrix of articular cartilage, based on Donnan equilibrium theory, and its use enables imaging of the human cadaveric metacarpal phalangeal joint.

RGS4 inhibits angiotensin II signaling and macrophage localization during renal reperfusion injury independent of vasospasm.

Vascular inflammation is a major contributor to the severity of acute kidney injury. In the context of vasospasm-independent reperfusion injury we studied the potential anti-inflammatory role of the Gα-related RGS protein, RGS4. Transgenic RGS4 mice were resistant to 25 min injury, although post-ischemic renal arteriolar diameter was equal to the wild type early after injury. A 10 min unilateral injury was performed to study reperfusion without vasospasm. Eighteen hours after injury, blood flow was decreased in the inner cortex of wild-type mice with preservation of tubular architecture. Angiotensin II levels in the kidneys of wild-type and transgenic mice were elevated in a sub-vasoconstrictive range 12 and 18 h after injury. Angiotensin II stimulated pre-glomerular vascular smooth muscle cells (VSMCs) to secrete the macrophage chemoattractant RANTES, a process decreased by angiotensin II R2 (AT2) inhibition. However, RANTES increased when RGS4 expression was suppressed implicating Gα protein activation in an AT2-RGS4-dependent pathway. RGS4 function, specific to VSMC, was tested in a conditional VSMC-specific RGS4 knockout showing high macrophage density by T2 MRI compared with transgenic and non-transgenic mice after the 10 min injury. Arteriolar diameter of this knockout was unchanged at successive time points after injury. Thus, RGS4 expression, specific to renal VSMC, inhibits angiotensin II-mediated cytokine signaling and macrophage recruitment during reperfusion, distinct from vasomotor regulation.

Delayed contrast-enhanced MRI of cartilage: comparison of nonionic and ionic contrast agents.

The objective of this study was to evaluate if cartilage fixed charge density is the only factor determining the distribution of the measured delayed gadolinium-enhanced magnetic resonance imaging of cartilage index, T(1) (Gd-DTPA(2-) ), across cartilage in the clinical delayed gadolinium-enhanced magnetic resonance imaging of cartilage protocol. Nineteen subjects with osteoarthritis and 14 controls were included. Cartilage T(1) (Gd) was measured following administration of 0.2 mmol kg(-1) of nonionic (Gd-DTPA-BMA) and, at a different date, anionic (Gd-DTPA(2-). T(1) (Gd-DTPA-BMA) was plotted against T(1) (Gd-DTPA(2-); a slope of 0 would indicate domination by charge effects; a nonzero slope would suggest that other factors influence T(1) (Gd-DTPA-BMA), and hence potentially T(1) (Gd-DTPA(2-). The low slope of the curve found in osteoarthritis subjects (0.31) indicates that Gd-DTPA-BMA penetrated most osteoarthritis cartilage to the same extent, and T(1) (Gd-DTPA-BMA) did not differentiate cartilages, which were differentiated by T(1) (Gd-DTPA(2-). The higher slopes in control subjects (0.88) are possibly due to inhibited transport of contrast agent into healthier cartilage, potentially exaggerated by the fast body clearance of the nonionic contrast agent. Overall, the use of anionic Gd-DTPA(2-) for delayed gadolinium-enhanced magnetic resonance imaging of cartilage is indicated for better discrimination of the health status of cartilage. Future studies could be designed to use contrast-enhanced dynamics to understand the transport properties of tissues in the joint and to evaluate the concentration of tissue constituents.

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.

Effects of chondrogenic and osteogenic regulatory factors on composite constructs grown using human mesenchymal stem cells, silk scaffolds and bioreactors.

Human mesenchymal stem cells (hMSCs) isolated from bone marrow aspirates were cultured on silk scaffolds in rotating bioreactors for three weeks with either chondrogenic or osteogenic medium supplements to engineer cartilage- or bone-like tissue constructs. Osteochondral composites formed from these cartilage and bone constructs were cultured for an additional three weeks in culture medium that was supplemented with chondrogenic factors, supplemented with osteogenic factors or unsupplemented. Progression of cartilage and bone formation and the integration between the two regions were assessed by medical imaging (magnetic resonance imaging and micro-computerized tomography imaging), and by biochemical, histological and mechanical assays. During composite culture (three to six weeks), bone-like tissue formation progressed in all three media to a markedly larger extent than cartilage-like tissue formation. The integration of the constructs was most enhanced in composites cultured in chondrogenic medium. The results suggest that tissue composites with well-mineralized regions and substantially less developed cartilage regions can be generated in vitro by culturing hMSCs on silk scaffolds in bioreactors, that hMSCs have markedly higher capacity for producing engineered bone than engineered cartilage, and that chondrogenic factors play major roles at early stages of bone formation by hMSCs and in the integration of the two tissue constructs into a tissue composite.

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.

Diffusion tensor imaging of native and degenerated human articular cartilage.

Diffusion tensor imaging (DTI) is potentially sensitive to collagen degeneration in cartilage. In this study, DTI was measured on human cartilage samples with interventions of trypsin and collagenase. The measured preferred diffusion direction was consistent with the zonal structure of collagen network. The glycosaminoglycan concentration decreased and apparent diffusion coefficient increased with both interventions. The fractional anisotropy (FA) was not affected by trypsin and showed a slight increase with combined trypsin and collagenase intervention. DTI in cartilage is technically challenging due to the low FA and the almost undetectable change with collagen disruption seen here.

Bone and cartilage tissue constructs grown using human bone marrow stromal cells, silk scaffolds and rotating bioreactors.

Human bone marrow contains a population of bone marrow stromal cells (hBMSCs) capable of forming several types of mesenchymal tissues, including bone and cartilage. The present study was designed to test whether large cartilaginous and bone-like tissue constructs can be selectively engineered using the same cell population (hBMSCs), the same scaffold type (porous silk) and same hydrodynamic environment (construct settling in rotating bioreactors), by varying the medium composition (chondrogenic vs. osteogenic differentiation factors). The hBMSCs were harvested, expanded and characterized with respect to their differentiation potential and population distribution. Passage two cells were seeded on scaffolds and cultured for 5 weeks in bioreactors using osteogenic, chondrogenic or control medium. The three media yielded constructs with comparable wet weights and compressive moduli ( approximately 25 kPa). Chondrogenic medium yielded constructs with higher amounts of DNA (1.5-fold) and glycosaminoglycans (GAG, 4-fold) per unit wet weight (ww) than control medium. In contrast, osteogenic medium yielded constructs with higher dry weight (1.6-fold), alkaline phosphatase (AP) activity (8-fold) and calcium content (100-fold) per unit ww than control medium. Chondrogenic medium yielded constructs that were weakly positive for GAG by contrast-enhanced MRI and alcian blue stain, whereas osteogenic medium yielded constructs that were highly mineralized by microCT and von Kossa stain. Engineered bone constructs were large (8mm diameter x 2mm thick disks) and resembled trabecular bone with respect to structure and mineralized tissue volume fraction (12%).