Dermatopathologic features of cutaneous squamous cell carcinoma and actinic keratosis: Consensus criteria and proposed reporting guidelines.

BACKGROUND: There is considerable variation in the literature regarding the dermatopathologic diagnostic features of and reporting guidelines for actinic keratosis (AK) and cutaneous squamous cell carcinoma (cSCC). OBJECTIVE: To develop consensus recommendations regarding diagnostic criteria, nomenclature, and reporting of AK and cSCC. METHODS: Literature review and cross-sectional multiround Delphi process including an international group of expert dermatopathologists followed by a consensus meeting. RESULTS: Consensus was achieved regarding the key dermatopathologic features necessary for diagnosing cSCC, AK, and associated variants; grading of degree of cellular differentiation in cSCC; utility of immunohistochemistry for diagnosis of cSCC; and pathologic features that should be reported for cSCC and AK. LIMITATIONS: Consensus was not achieved on all questions considered. CONCLUSION: Despite the lack of clarity in the literature, there is consensus among expert dermatopathologists regarding diagnostic criteria and appropriate reporting of AK and cSCC. Widespread implementation of these consensus recommendations may improve communication between dermatopathologists and clinicians, facilitating appropriate treatment of AK and cSCC.

Emerging roles for ectodomain shedding in the regulation of inflammatory responses.

The multistep model of leukocyte recruitment to sites of inflammation has helped elucidate specific molecular cues for each of the individual steps. However, it is less clear how cells transition between the different steps and how the complex interactions are coordinately regulated. Once a leukocyte sticks to the endothelium, it only takes a few minutes to reach the subendothelial basement membrane, so the transitions and regulatory mechanisms must be rapid. We put forward the hypothesis that proteolytic shedding of cell surface proteins provides a mechanism to aid in the rapid transition of cells and coordinate the complex, multistep process of leukocyte recruitment in response to inflammatory stimuli. Support for this hypothesis is provided from analyses of disease states and from studies with protease inhibitors and genetically engineered mutations that prevent "ectodomain shedding" of cell surface proteins and consequently perturb the inflammatory response.

ADAM10 mediates ectodomain shedding of the betacellulin precursor activated by p-aminophenylmercuric acetate and extracellular calcium influx.

Betacellulin belongs to the family of epidermal growth factor-like growth factors that are expressed as transmembrane precursors and undergo proteolytic ectodomain shedding to release a soluble mature growth factor. In this study, we investigated the ectodomain shedding of the betacellulin precursor (pro-BTC) in conditionally immortalized wild-type (WT) and ADAM-deficient cell lines. Sequential ectodomain cleavage of the predominant cell-surface 40-kDa form of pro-BTC generated a major (26-28 kDa) and two minor (20 and 15 kDa) soluble forms and a cellular remnant lacking the ectodomain (12 kDa). Pro-BTC shedding was activated by calcium ionophore (A23187) and by the metalloprotease activator p-aminophenylmercuric acetate (APMA), but not by phorbol esters. Culturing cells in calcium-free medium or with the protein kinase Cdelta inhibitor rottlerin, but not with broad-based protein kinase C inhibitors, blocked A23187-activated pro-BTC shedding. These same treatments were without effect for constitutive and APMA-induced cleavage events. All pro-BTC shedding was blocked by treatment with a broad-spectrum metalloprotease inhibitor (GM6001). In addition, constitutive and activated pro-BTC shedding was differentially blocked by TIMP-1 or TIMP-3, but was insensitive to treatment with TIMP-2. Pro-BTC shedding was functional in cells from ADAM17- and ADAM9-deficient mice and in cells overexpressing WT or catalytically inactive ADAM17. In contrast, overexpression of WT ADAM10 enhanced constitutive and activated shedding of pro-BTC, whereas overexpression of catalytically inactive ADAM10 reduced shedding. These results demonstrate, for the first time, activated pro-BTC shedding in response to extracellular calcium influx and APMA and provide evidence that ADAM10 mediates constitutive and activated pro-BTC shedding.

A disintegrin and metalloproteinase 10-mediated cleavage and shedding regulates the cell surface expression of CXC chemokine ligand 16.

CXC chemokine ligand (CXCL)16 and scavenger receptor for phosphatidylserine and oxidized low-density lipoprotein were independently identified as a chemokine and a scavenger receptor, respectively, but have since been shown to be identical. CXCL16 is synthesized as a transmembrane protein with its chemokine domain at the end of a mucin-rich stalk. When expressed at the cell surface, CXCL16 functions as a scavenger receptor, binding and internalizing oxidized low-density lipoprotein and bacteria. As a soluble form, CXCL16 is a chemoattractant for activated CD4+ and CD8+ T cells through binding its receptor, CXCR6. In this study, we examined the mechanisms that regulate the conversion between these two functionally distinct forms of CXCL16. We demonstrate that murine CXCL16 is synthesized as an intracellular precursor that is rapidly transported to the cell surface where it undergoes metalloproteinase-dependent cleavage, causing the release of a fragment that constitutes the majority of the CXCL16 extracellular domain. Using a novel retroviral system for the generation of short interfering RNAs, we show that knockdown of a disintegrin and metalloproteinase (ADAM) family protease ADAM10 decreases this constitutive shedding of CXCL16. Furthermore, we show that overexpression of ADAM10 increases CXCL16 shedding, whereas overexpression of a dominant-negative form of ADAM10 lowers shedding of CXCL16 in a similar manner to short interfering RNAs. Through the modulation of ADAM10 function, we demonstrate that ADAM10-mediated constitutive shedding is a key regulator of CXCL16 cell surface expression. The identification of ADAM10 as a major protease responsible for the conversion of CXCL16 from a membrane-bound scavenger receptor to a soluble chemoattractant will provide new information for understanding the physiological function of this molecule.

Stimulated shedding of vascular cell adhesion molecule 1 (VCAM-1) is mediated by tumor necrosis factor-alpha-converting enzyme (ADAM 17).

A variety of cell surface adhesion molecules can exist as both transmembrane proteins and soluble circulating forms. Increases in the levels of soluble adhesion molecules have been correlated with a variety of inflammatory diseases, suggesting a pathological role. Although soluble forms are thought to result from proteolytic cleavage from the cell surface, relatively little is known about the proteases responsible for their release. In this report we demonstrate that under normal culture conditions, cells expressing vascular cell adhesion molecule 1 (VCAM-1) release a soluble form of the extracellular domain that is generated by metalloproteinase-mediated cleavage. VCAM-1 release can be rapidly simulated by phorbol 12-myristate 13-acetate (PMA), and this induced VCAM-1 shedding is mediated by metalloproteinase cleavage of VCAM-1 near the transmembrane domain. PMA-induced VCAM-1 shedding occurs as the result of activation of a specific pathway, as the generation of soluble forms of three other adhesion molecules, E-selectin, platelet-endothelial cell adhesion molecule 1, and intercellular adhesion molecule 1, are not altered by PMA stimulation. Using cells derived from genetically deficient mice, we identify tumor necrosis factor-alpha-converting enzyme (TACE or ADAM 17) as the protease responsible for PMA-induced VCAM-1 release, including shedding of endogenously expressed VCAM-1 by murine endothelial cells. Therefore, TACE-mediated shedding of VCAM-1 may be important for the regulation of VCAM-1 function at the cell surface.

An NF-kappaB-dependent transcriptional program is required for collagen remodeling by human smooth muscle cells.

Although remodeling of vessels can dramatically alter lumen diameter and clinical sequelae, the molecular mechanisms regulating extracellular matrix turnover and remodeling are still not well understood. To investigate these processes in human smooth muscle, we have compared their culture on monomer and polymerized collagen gels, conditions that mimic some of the features of injured and normal vessels, respectively. We show that culture on polymerized, but not monomer, collagen leads to the activation of the transcription factor NF-kappaB through phosphorylation and degradation of its inhibitor, IkappaBalpha. Coincident with NF-kappaB activation, expression of MMP1, MMP2, and alpha2 integrin increases on polymerized collagen. Specific inhibition of NF-kappaB by retroviral overexpression of wild-type IkappaBalpha or phosphorylation-resistant, IkappaBalpha-stabilized mutant (IkappaBalphaSer32,36/Ala) reverses the increases in MMP1 and alpha2beta1 on polymerized collagen and decreases collagen gel contraction and degradation. However, forced overexpression of alpha2beta1 integrin or MMP1 in smooth muscle cells expressing IkappaBalphaSer32,36/Ala rescues their ability to contract collagen gels. Thus, polymerized collagen induces NF-kappaB-dependent expression of MMP1 and alpha2beta1 integrin, that are required for smooth muscle extracellular matrix remodeling.

Efficient expression of exogenous genes in primary vascular cells using IRES-based retroviral vectors.

Analysis of gene function in primary vascular cells has been particularly limited by low transfection efficiencies. Using internal ribosomal entry site (IRES)-based retroviral vectors, we demonstrate efficient infection (range of 45%-95%) of primary human endothelial and smooth muscle cells with genes varying in size from 1.3 to 4.5 kb. Because IRES vectors are designed to allow the expression of two genes from a single mRNA, we can show excellent correlation between the expression of a reporter gene and an inserted gene of interest. Reporter gene expression allows rapid (24-48 h) and unambiguous identification of transduced cells. Additionally, reporter gene expression can be used to isolate subpopulations of cells that express distinct levels of cistron 1 genes by flow cytometry, and sorted cells maintain relative levels of gene expression over multiple passages in culture. Two examples of the usefulness of these vectors to characterize gene function in primary vascular cells include (i) the inhibition of endothelial cell inflammatory responses in a polyclonal population by the expression of a dominant negative inhibitor of nuclear factor-kappaB and (ii) monitoring the in vitro evolution of smooth muscle cells provided with a selective growth advantage by transduction with telomerase. Potential applications of retroviral expression strategies in vascular biology are also discussed.