c-Src/Cav1-dependent activation of the EGFR by Dsg2.

The desmosomal cadherin, desmoglein 2 (Dsg2), is deregulated in a variety of human cancers including those of the skin. When ectopically expressed in the epidermis of transgenic mice, Dsg2 activates multiple mitogenic signaling pathways and increases susceptibility to tumorigenesis. However, the molecular mechanism responsible for Dsg2-mediated cellular signaling is poorly understood. Here we show overexpression as well as co-localization of Dsg2 and EGFR in cutaneous SCCs in vivo. Using HaCaT keratinocytes, knockdown of Dsg2 decreases EGFR expression and abrogates the activation of EGFR, c-Src and Stat3, but not Erk1/2 or Akt, in response to EGF ligand stimulation. To determine whether Dsg2 mediates signaling through lipid microdomains, sucrose density fractionation illustrated that Dsg2 is recruited to and displaces Cav1, EGFR and c-Src from light density lipid raft fractions. STED imaging confirmed that the presence of Dsg2 disperses Cav1 from the cell-cell borders. Perturbation of lipid rafts with the cholesterol-chelating agent MβCD also shifts Cav1, c-Src and EGFR out of the rafts and activates signaling pathways. Functionally, overexpression of Dsg2 in human SCC A431 cells enhances EGFR activation and increases cell proliferation and migration through a c-Src and EGFR dependent manner. In summary, our data suggest that Dsg2 stimulates cell growth and migration by positively regulating EGFR level and signaling through a c-Src and Cav1-dependent mechanism using lipid rafts as signal modulatory platforms

Enhancement of cutaneous wound healing by Dsg2 augmentation of uPAR secretion

In addition to playing a role in adhesion, desmoglein 2 (Dsg2) is an important regulator of growth and survival signaling pathways, cell proliferation, migration and invasion, and oncogenesis. While low-level Dsg2 expression is observed in basal keratinocytes and is downregulated in non-healing venous ulcers, overexpression has been observed in both melanomas and non-melanoma malignancies. Here, we show that transgenic mice overexpressing Dsg2 in basal keratinocytes primed the activation of mitogenic pathways, but did not induce dramatic epidermal changes or susceptibility to chemical-induced tumor development. Interestingly, acceleration of full-thickness wound closure and increased wound-adjacent keratinocyte proliferation was observed in these mice. As epidermal cytokines and their receptors play critical roles in wound healing, Dsg2-induced secretome alterations were assessed with an antibody profiler array and revealed increased release and proteolytic processing of the urokinase-type plasminogen activator receptor (uPAR). Dsg2 induced uPAR expression in the skin of transgenic compared to wild-type mice. Wound healing further enhanced uPAR in both epidermis and dermis with concomitant increase in the pro-healing laminin-332, a major component of the basement membrane zone, in transgenic mice. This study demonstrates that Dsg2 induces epidermal activation of various signaling cascades and accelerates cutaneous wound healing, in part, through uPAR-related signaling cascades

Cell Cycle- and Cancer-Associated Gene Networks Activated by Dsg2: Evidence of Cystatin A Deregulation and a Potential Role in Cell-Cell Adhesion

This work was supported by grants from the National Institutes of Health (Mahoney, R01AR056067; Riobo, RO1 GM088256). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

ZFP36L1 Negatively Regulates Erythroid Differentiation of CD34+ Hematopoietic Stem Cells by Interfering with the Stat5b Pathway

ZFP36L1 negatively regulates erythroid differentiation of human hematopoietic progenitors by directly binding the 3′ UTR of Stat5b mRNA, thereby triggering its degradation. This study shows that posttranscriptional regulation is involved in the control of hematopoietic differentiation

DSG2 synergizes with hedgehog signaling to promote skin tumor development

Basal cell and squamous cell carcinomas (BCC, SCC) are the most commonly diagnosed cancers in the United States. BCC are the result of aberrant activation of the canonical Hedgehog (Hh) pathway, typically via loss of function of the tumor suppressor Patched1 resulting from UV exposure. Hh signaling in SCC is less well understood. Treatment for both SCC and BCC is typically surgical resection; however, more advanced disease requires alternative approaches. Vismodegib, a small molecule inhibitor of the Hh pathway, is approved for use in advanced BCCs. However, major adverse effects and acquired drug resistance limit the effectiveness of this therapy. Identifying additional therapeutic targets may allow for more effective treatment. We previously reported that Desmoglein 2 (Dsg2), a desmosomal cadherin, is overexpressed in skin malignancies. Interestingly, Dsg2 expression corresponds with Gli1, a marker of canonical Hh activity, in both normal skin and tumors. Furthermore, we have demonstrated that Dsg2 enhances squamous tumor development and dysregulates signaling cascades that intersect with the Hh pathway. Here we cross the Ptc1+/lacZ reporter mouse, a model that mimics the familial form of BCC (Gorlin syndrome), with transgenic mouse models overexpressing Dsg2 in differential cell compartments of the epidermis, to assess the role of Hh-Dsg2 crosstalk in BCC and SCC development. Our results indicate that Dsg-Hh pathway interplay has a limited role in SCC development, but demonstrates that Hh and Dsg2 synergize to promote BCC formation. Immunohistochemical analysis of lesions, combined with in vitro approaches, implicate Stat3 as a mediator of this effect. Finally, we establish that concurrent inhibition of both Stat3 and Hh pathways is more effective at reducing Gli1 expression and tumor cell viability than targeting either pathway individually. The results here have implications for the clinical treatment of BCC, and by extension other Hh-dependent malignancies in which Stat3 signaling is deregulated

Loss of CSTA leads to destabilized intercellular connections.

<p>Cells were treated with non-targeting pool scrRNA or with <i>CSTA</i> siRNA (CSTA KD) followed by mechanical stretching for 4 hr. Cells were allowed to adhere, fixed, and immunostained for Dsg2 (A) and cytokeratin 14 (B) or lysed in Laemmli buffer and immunoblotted for desmoplakin (C). Knockdown of CSTA in keratinocytes resulted in cytoplasmic relocalization of Dsg2, breakage of cytokeratin intercellular connections, and loss of the desmosomal protein, desmoplakin.</p

Top ten genes up- or down-regulated in response to Dsg2.

<p>Top ten genes up- or down-regulated in response to Dsg2.</p

Differential gene expression between Inv-Dsg2 transgenic and wild-type mouse skin.

<p>(A) Total RNA was isolated from the skin of 2 wild-type and 2 Inv-Dsg2 transgenic mice, reverse transcribed, biotin-labeled and applied to a mouse cDNA microarray. The dendogram (heat map) shows that 492 genes were either up-regulated (red/orange) or down-regulated (blue/green) in transgenic (T1 and T2) and control (W1 and W2) mice. (B) Volcano plot shows the log2 (fold change) in x-axis versus the—log10 (p value) in the y-axis. The points having a fold-change less than 2 (log2 = 1) are shown in gray. The vertical green lines demarcate where the fold change equals 2 (right line) or equals—2 (left line). The horizontal green line demarcates where the p value is 0.05, with points above the line having p<0.05 and points below the line having p>0.05. Depicted in red are the genes that exhibit a greater than 2 fold change with a p>0.05 in transgenic epidermis as compared to control. The arrows indicate genes of interest. (C) Quantitative real-time RT-PCR analysis reveals an average of 34.33±1.32 fold increase in Dsg2 RNA expression in Inv-Dsg2 transgenic (Tg) compared to that of wild-type (WT). In addition, RNA expression for transgenic relative to control were: <i>Csta1</i>, 113.24 ±2.23; <i>Csta2</i>, 1227.04 ±1.26; <i>Csta2l1</i>, 1.11 ±0.47; <i>Csta3</i>, 26.97±0.44 (Bar = mean ± s.d.; (*p< 0.05; **p<0.01; ***p<0.001; Student’s <i>t</i> test).</p

Dsg2 enhances cystatin A expression <i>in vivo</i>.

<p>(A) Western blot analysis of skin lysates from 3 newborn and 3 adult C57Bl6 mice shows high expression of Csta in newborn but virtually undetectable in adult skin. Actin was used as a control for equal loading. (B) Immunofluorescent staining confirms the Western blotting results showing high level of Csta in newborn wild-type mouse skin. Enlarged image in inset shows cytoplasmic as well as nuclear staining for Csta. (C) Western analysis for Dsg2 and Csta in adult wild-type and Inv-Dsg2 transgenic mouse skin. The results showed expression of the Flag-tagged Dsg2 and Csta in the transgenic but not wild-type mice. Actin showed equal loading. (D) Immunofluorescence was performed on adult skin of wild-type and transgenic mice revealing increased levels CSTA in transgenic skin. Nuclei were counter-stained with DAPI (blue).</p

Upregulation of cystatin A, S100 and cyclin genes in Dsg2 transgenic mice.

<p>Upregulation of cystatin A, S100 and cyclin genes in Dsg2 transgenic mice.</p