CLIC4 (Chloride intracellular channel 4) is a member of a family of intracellular chloride channel proteins that are ubiquitously expressed in multiple tissue types. In keratinocytes and other cell types, cytoplasmic CLIC4 translocates to the nucleus under conditions of metabolic stress, growth arrest, apoptosis and DNA damage. Nuclear targeting of CLIC4 to basal undifferentiated keratinocytes causes cell cycle arrest and terminal differentiation. CLIC4 is excluded from the nucleus in epithelial cancer cells but upregulated in tumor stroma. CLIC4 is a direct downstream effector of p53, c-Myc and PPAR beta. Nuclear translocation of CLIC4 is defective in cancer cells. Thus, understanding the control of nuclear trafficking in normal cells is essential to dissect the defect in cancer cells. Nitric oxide directly enhances nuclear translocation of CLIC4 and modifies it on specific cysteine residue(s) by S-nitrosylation. NO-induced S-nitrosylation enhances association of wildtype CLIC4 with the nuclear import proteins Ran, importin alpha and NTF2. Preliminary molecular modeling in collaboration with Jinhui Ding show cysteines 35 and 234 in close proximity to each other in the folded protein and nitrosylation of cysteine 234 may induce conformational changes to link cysteine 234 to cysteine 35 through two water molecules. Mutations targeted at all four cysteine residues show that cysteine 35 is essential for protein stability and mutation of the thiol (to alanine or serine) leads to proteasome-mediated degradation. Remarkably, cysteine 35 also seems to regulate S-nitrosylation of the protein as mutants (alanine and serine) of cysteine 35 show hyper S-nitrosylation even in untreated cells. This increased S-nitrosylation likely occurs at cysteine 234 as a double mutant (cysteine to serine) at positions 35 and 234 shows no S-nitrosylation. In addition, the mutation of cysteine 35 results in marked nuclear distribution that is likely a consequence of its enhanced association with the nuclear import proteins Ran and importin alpha. Thus cysteine 35 may be an allosteric or catalytic regulator of S-nitrosylation and the subcellular distribution of CLIC4. Our current results suggest that both iNOS and eNOS participate in nitrosylation of CLIC4 critical cysteines and changes in redox state in cancer cells and particularly these enzyme systems could alter potential for nuclear localization of CLIC4 in tumors. We have recently discovered that TGFbeta enhances the expression and nuclear translocation of CLIC4 in association with Schnurri-2 in keratinocytes. In turn, nuclear CLIC4 enhances TGFbeta signaling, independent of Schnurri-2. Nuclear CLIC4 enhances TGFbeta signal transduction by increasing the levels of phospho-Smad2, a key mediator of the TGFbeta response. CLIC4 physically interacts with phospho-Smad2 and prevents phosphatase mediated dephosphorylation of Smad2, thereby enhancing and prolonging the TGFbeta signal. Nuclear CLIC4 specifically inhibits the interaction of phospho-Smad2 with PPM1a which is the only known Smad phosphatise. CLIC4 may however also interact with other hitherto undetected Smad phosphatases. CLIC4 is strongly associated with myofibroblast conversion in the stroma of multiple tumor types. CLIC4 overexpression in primary mouse dermal fibroblasts enhances the expression of alpha smooth muscle actin. Fibroblast transdifferentiation by TGFbeta depends on ERK activation. CLIC4 overexpression enhances phosphorylation of ERK and synergizes with TGFbeta in activating ERK. These data show that CLIC4 may participate in the stromal reaction to cancer by virtue of its role as a TGFbeta pathway modifier. We are currently at the chimeric stage in the development of skin targeted CLIC4 null mouse using recombineering methods. The epidermal growth factor receptor (EGFR, HER1, ErbB1) is activated by mutation, amplification or ligand over-expression in skin carcinomas. It was discovered that cutaneous folliculitis is a dose limiting toxicity of systemic inhibition of EGFR for cancer therapy. Similar lesions were detected in the skin of mice where EGFR has been genetically deleted, and we have used keratinocytes from these mice, and their ras oncogene transformed derivatives, to identify critical pathways involved in the inflammatory response. Microarray analysis performed on cDNA from ras transformed mouse keratinocytes derived from wildtype or EGFR null mice or treated with EGFR tyrosine kinase inhibitors AG1478, PD153035, or Tarceva generated a 25 gene signature for EGFR ablation. Pathway analysis of the EGFR signature demonstrated the possible involvement of p38 in EGFR ablation. In ras infected keratinocytes, p38 mediated inhibition of ERK1/2 is lost, which also occurs in cells from head and neck squamous cell carcinomas. In EGFR ablated samples total p38, and p38 alpha are unchanged where as p38 delta is up-regulated. Further studies on p38 delta should clarify the involvement of this isoform in toxicity to EGFR inhibitors. Cytokine induced expression of Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF) is dependent upon EGFR activity in human and mouse keratinocytes. A mouse model where EGFR is ablated only in the skin develops skin inflammation characterized by cycles of hair growth and hair loss. EGFR conditional knockout mice display higher numbers of circulating neutrophils (12 fold), eosinophils (23 fold) and basophils (4 fold) relative to their single transgenic littermates. No differences were observed in lymphocyte counts. CD45 antigen is increased in the skin tissue sections of the conditional null mice. Cutaneous leukocyte abundance is further increased upon painting the dorsal skin of the mice with TPA. While transcripts for a subset of inflammatory mediators (MIP-2, MIP-3alpha, MCP-1 and GM-CSF) are elevated in the total skin extracts of ablated mice, epidermal GM-CSF is lower. These data suggest that in the absence of a functional EGFR pathway, the proinflammatory milieu in the skin cannot fully induce the expression of GM-CSF in keratinocytes, while the leukocyte populations retain GM-CSF expression during the inflammatory response causing the overall increase of this cytokine in total skin extracts. In the skin of 3 hr post partum mice through 5 months of age, mast cells significantly increase in the absence of epidermal EGFR starting from day 7. By the third week of age these mice display pruritus, and it is likely that the increased mast cell component in their skin is linked to the worsening of their skin condition. Furthermore, plasma samples obtained from young and older conditional EGFR null or control littermates defined a Th17 prone environment. In the EGFR conditional knockouts higher levels of IL-17, IL-6 and IP-10 (CXCL-10) were measured in both acute and chronic conditions. Additional changes in circulating chemokines distinguished the EGFR conditional null mice for their control littermates indicating the global scope of the immunological abnormalities that epidermal ablation of the EGFR can cause at the systemic level. We anticipate that many of these changes will be translatable to the adverse skin phenotype in cancer patients receiving anti-EGFR therapy. In collaboration with Dolph Hatfield, we are studying two genetically altered mouse strain [summary truncated at 7800 characters]
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