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Fibroblast differentiation into myofibroblasts is a key event during normal wound repair. We have previously demonstrated an age-related defect in this process associated with impaired synthesis of hyaluronan (HA) synthase (HAS) 2 but failed to prescribe its role in a mechanistic sense. Here we demonstrate that in addition to HAS2, there is loss of EGF receptor (EGF-R) in aged cells, and both are required for normal fibroblast functionality. Analysis of molecular events revealed that in young cells, transforming growth factor (TGF)-beta1-dependent phenotypic activation uses two distinct but cooperating pathways that involve TGF-beta receptor/Smad2 activation and EGF-mediated EGF-R/extracellular signal-regulated kinase (ERK) 1/2 signaling, and the latter is compromised with in vitro aging. Pharmacological inhibition of any of the five intermediates (TGF-beta receptor, Smad2, EGF, EGF-R, and ERK1/2) attenuated TGF-beta1 induction of alpha-smooth muscle actin. We present evidence that the HA receptor CD44 co-immunoprecipitates with EGF-R after activation by TGF-beta1. This interaction is HA-dependent because disruption of HA synthesis abrogates this association and inhibits subsequent ERK1/2 signaling. In aged fibroblasts, this association is lost with resultant suppression of ERK1/2 activation. Forced overexpression of EGF-R and HAS2 in aged cells restored TGF-beta1-mediated HA-CD44/EGF-R association and alpha-smooth muscle actin induction. Taken together, these results demonstrate that HA can serve as a signal integrator by facilitating TGF-beta1-mediated CD44-EGF-R-ERK interactions and ultimately fibroblast phenotype. We propose a model to explain this novel mechanism and the functional consequence of age-dependent dysregulation.

Adult bone marrow multipotential stromal cells (MSCs) hold great promise in regenerative medicine and tissue engineering. However, due to their low numbers upon harvesting, MSCs need to be expanded in vitro without biasing future differentiation for optimal utility. In this concept paper, we focus on the potential use of epidermal growth factor (EGF), prototypal growth factor for enhancing the harvesting and/or differentiation of MSCs. Soluble EGF was shown to augment MSC proliferation while preserving early progenitors within MSC population, and thus did not induce differentiation. However, tethered form of EGF was shown to promote osteogenic differentiation. Soluble EGF was also shown to increase paracrine secretions including VEGF and HGF from MSC. Thus, soluble EGF can be used not only to expand MSC in vitro, but also to enhance paracrine secretion through drug-releasing MSC-encapsulated scaffolds in vivo. Tethered EGF can also be utilized to direct MSC towards osteogenic lineage both in vitro and in vivo.

Publisher Summary This chapter provides an understanding of the calpain family of intracellular cysteine proteases. Earlier work focused on in vitro regulation and activities of these proteases, primarily the two ubiquitous isoforms μ-calpain (calpain 1) and m-calpain (calpain 2). Ongoing structure–function studies have culminated in identification of the molecular structure of the two ubiquitous calpains, and are now deciphering how these enzymes might be activated and modulated, and how they interact with modifiers and targets. Mechanisms to reduce the requirement for calcium to the physiological range have been proposed. These include phospholipid binding, release of calpain from its inhibitor calpastatin, and binding of activator proteins. Evidence supports a critical role for calpain as a signaling intermediate downstream of both integrin and growth factor signaling pathways. The critical importance of the ubiquitous m- and μ-calpain in normal development is demonstrated by transgenic mice deficient in the regulatory subunit that eliminates detectable calpain activity proliferation. Initial studies of the role of calpain were conducted in platelets, an interesting system where calpain clearly plays a role in secretion, adhesion, and aggregation.

We tracked the extracellular fate of proteins of pulmonary origin using the technique of stable isotope labeling of amino acids in cell culture (SILAC) in cell-impermeable Transwell culture systems. We find that irradiation to murine lung and lung-derived cells induces their release of proteins that are capable of entering neighboring cells, including primary murine bone marrow cells as well as prostate cancer and hematopoietic cell lines. The functional classification of transferred proteins was broad and included transcription factors, mediators of basic cellular processes and components of the nucleosome remodeling and deacetylase complex, including metastasis associated protein 3 and retinoblastoma-binding protein 7. In further analysis we find that retinoblastoma-binding protein 7 is a transcriptional activator of E-cadherin and that its intercellular transfer leads to decreased gene expression of downstream targets such as N-cadherin and vimentin. SILAC-generated data sets offer a valuable tool to identify and validate potential paracrine networks that may impact relevant biologic processes associated with phenotypic and genotypic signatures of health and disease.

The ubiquitously expressed family of alpha-actinins bridges actin filaments to stabilize adhesions, a process disrupted during growth factor-induced migration of cells. During the dissolution of the actin cytoskeleton, actinins are phosphorylated on tyrosines, although the consequences of this are unknown. We expressed the two isoforms of human alpha-actinin in murine fibroblasts that express human epidermal growth factor receptor (EGFR) and found that both alpha-actinin 1 (ACTN1) and alpha-actinin 4 (ACTN4) were phosphorylated on tyrosine residues after stimulation with EGF, although ACTN4 was phosphorylated to the greater extent. This required the activation of Src protein-tyrosine kinase and p38-MAPK (and phosphoinositide trisphosphate kinase in part) but not MEK/ERK or Rac1, as determined by inhibitors. The EGF-induced phosphorylation sites of ACTN4 were mapped to tyrosine 4, the major site, and tyrosine 31, the minor one. Truncation mutagenesis showed that the C-terminal domains of ACTN4 (amino acids 300-911), which cross-link the actin binding head domains, act as an inhibitory domain for both actin binding and EGF-mediated phosphorylation. These two properties were mutually exclusive; removal of the C terminus enhanced actin binding of ACTN4 mutants while limiting EGF-induced phosphorylation, and conversely EGF-stimulated phosphorylation of ACTN4 decreased its affinity to actin. Interestingly, a phosphomimetic of tyrosine 265 (which can be found in carcinoma cells and lies near the K255E mutation that causes focal segmental glomerulosclerosis) demonstrated increased actin binding activity and susceptibility of ACTN4 to calpain-mediated cleavage; this variant also retarded cell spreading. Remarkably, either treatment of cells with low concentrations of latrunculin A, which has been shown to depolymerize F-actin, or the deletion of the actin binding domain (100-252 amino acids) of ACTN4Y265E restored EGF-induced phosphorylation. An F-actin binding assay in vitro showed that Y4E/Y31E, a mimetic of diphosphorylated ACTN4, bound F-actin slightly compared with wild type (WT). Importantly, the EGF-mediated phosphorylation of ACTN4 at tyrosine 4 and 31 significantly inhibited multinucleation of proliferating NR6WT fibroblasts that overexpress ACTN4. These results suggest that EGF regulates the actin binding activity of ACTN4 by inducing tyrosyl-directed phosphorylation.

Author Manuscript 2011 April 29., Epidermal growth factor receptor (EGFR)-mediated signaling helps regulate bone development and healing through its effects on osteogenic cells. Here, we show how EGFR activity and osteogenic differentiation responses in primary human bone marrow-derived multipotent stromal cells (MSCs) are influenced by presenting covalently tethered epidermal growth factor (tEGF) on the culture substratum, a presentation mode that reduces EGFR internalization and restricts signaling to the cell surface. In both absence and presence of tEGF, MSCs increase expression levels of EGFR and its heterodimerization partner HER2 during the course of osteogenic differentiation. tEGF substrata increased levels of phosphorylated EGFR and phosphorylated extracellular regulated kinase (ERK) compared to control substrata, and these elevations were associated with a twofold enhancement of MSC alkaline phosphatase activity at day 7 and matrix mineralization at day 21. Surprisingly, addition of soluble EGF (sEGF) to cells cultured on tEGF substrata reduces osteogenic differentiation, even though EGFR signaling is more strongly activated in acute, short-term manner by sEGF treatment than by tEGF treatment. A striking concomitant result of the sEGF effects is near-complete downregulation of EGFR and HER2, demonstrating that the tEGF/EGFR interaction is dynamically reversible even though temporally sustained. Taken together, our results show that enhanced MSC osteogenic differentiation corresponds to a sustained combination of receptor expression and ligand presentation, both of which are maintained by tEGF., National Institutes of Health (U.S.) (Grant R01-GM059870-07), National Institutes of Health (U.S.) (Grant R01-DE 019523-10), National Institutes of Health (U.S.) (Grant P40RR017447), United Negro College Fund (Merck Postdoctoral Fellowship), Georgia Institute of Technology (FACES Fellowship), National Center for Research Resources (U.S.) (Grant P40RR017447)

Strategies to combine aspirated marrow cells with scaffolds to treat connective tissue defects are gaining increasing clinical attention and use. In situations such as large defects where initial survival and proliferation of transplanted connective tissue progenitors (CTPs) are limiting, therapeutic outcomes might be improved by using the scaffold to deliver growth factors that promote the early stages of cell function in the graft. Signaling by the epidermal growth factor receptor (EGFR) plays a role in cell survival and has been implicated in bone development and homeostasis. Providing epidermal growth factor (EGF) in a scaffold-tethered format may sustain local delivery and shift EGFR signaling to pro-survival modes compared to soluble ligand. We therefore examined the effect of tethered EGF on osteogenic colony formation from human bone marrow aspirates in the context of three different adhesion environments using a total of 39 donors. We found that tethered EGF, but not soluble EGF, increased the numbers of colonies formed regardless of adhesion background, and that tethered EGF did not impair early stages of osteogenic differentiation., National Institute of General Medical Sciences (U.S.) (Grant NIH RO1 AR42997), National Institute of General Medical Sciences (U.S.) (Grant NIH RO1 AG024980), National Institute of General Medical Sciences (U.S.) (Grant NIH RO1 GM59870), National Institute of General Medical Sciences (U.S.) (Grant NIH DE019523)

Profilin-1 (Pfn1), a ubiquitously expressed actin-binding protein, has been regarded as a tumor-suppressor molecule for breast cancer. Since AKT signaling impacts cell survival and proliferation, in this study we investigated whether AKT activation in breast cancer cells is sensitive to perturbation of Pfn1 expression. We found that even a moderate overexpression of Pfn1 leads to a significant reduction in phosphorylation of AKT in MDA-MB-231 breast cancer cells. We further demonstrated that Pfn1 overexpression in MDA-MB-231 cells is associated with a significant reduction in the level of the phosphoinositide regulator of AKT, PIP(3), and impaired membrane translocation of AKT that is required for AKT activation, in response to EGF stimulation. Interestingly, Pfn1-overexpressing cells showed post-transcriptional upregulation of PTEN. Furthermore, when PTEN expression was silenced, AKT phosphorylation was rescued, suggesting PTEN upregulation is responsible for Pfn1-dependent attenuation of AKT activation in MDA-MB-231 cells. Pfn1 overexpression induced PTEN upregulation and reduced AKT activation were also reproducible features of BT474 breast cancer cells. These findings may provide mechanistic insights underlying at least some of the tumor-suppressive properties of Pfn1.

Signals that can mediate ligand-induced receptor internalization and calcium regulation are present in a 48-amino acid "calcium-internalization" domain in the C' terminus of the epidermal growth factor (EGF) receptor. The basis of calcium and internalization regulation signalled by this 48-amino acid sequence was analyzed using deletion and substitution mutant receptors. Cells expressing truncated receptors containing either the NH2- or COOH-terminal portion of the 48-residue domain displayed high affinity EGF-dependent endocytosis and receptor down-regulation. These endocytosis-competent EGF receptor mutants that lacked any autophosphorylation site were unable to increase the concentration of intracellular calcium. To investigate the role of self-phosphorylation in EGF-induced calcium mobilization, phenylalanine was substituted for the single autophosphorylated tyrosine residue in this region of an internalization-competent truncated receptor. The receptor-mediated calcium response was abolished, while ligand-dependent receptor internalization was unimpaired. These results demonstrate that EGF-dependent receptor endocytosis and calcium mobilization are separate events. Tyrosine self-phosphorylation is required for increased [Ca2+]i, while structural features distinct from autophosphorylation are required for receptor internalization.

Activation of the EGF receptor tyrosine kinase by ligand indirectly activates a series of other cellular enzymes, including protein kinase C. To test the hypothesis that phosphorylation of the EGF receptor by protein kinase C provides an intracellular negative feedback loop to attenuate EGF receptor signaling, we used scanning EM to follow the characteristic EGF-induced retraction of lamellipodia and concomitant cell shape changes. Wild type and mutant EGF receptors were expressed in receptor-deficient NR6 cells. The mutant receptors were prepared by truncation at C' terminal residue 973 (c'973) to provide resistance to ligand-induced down regulation that strongly attenuates receptor signaling and by replacement of threonine 654 (T654) with alanine (A654) to remove the site of phosphorylation by protein kinase C. Cells expressing WT and c'973 EGF receptors demonstrated characteristic lamellipodial retraction after exposure to EGF, with the non-down regulating c'973 EGF receptors responding more rapidly. Exposure of cells to TPA blocked this response. Replacement of T654 by alanine resulted in EGF receptors that were resistant to TPA. Cells expressing the A654 mutation underwent more rapid and more extensive morphologic changes than cells with the corresponding T654 EGF receptor. In cells expressing T654 EGF receptors, down regulation of protein kinase C resulted in more rapid and extensive EGF-induced changes similar to those seen in cells expressing A654 EGF receptors. These data indicate that activation of protein kinase C and subsequent phosphorylation of the EGF receptor at T654 lead to rapid physiological attenuation of EGF receptor signaling.

Experiments undertaken to investigate the binding of epidermal growth factor by hepatocyte nuclei showed that: (a) isolated nuclei from both normal and regenerating rat liver are capable of binding 125I-epidermal growth factor, (b) the nuclear epidermal growth factor-binding protein is similar in molecular weight to the plasma membrane epidermal growth factor receptor, (c) monoclonal antibodies produced against the plasma membrane epidermal growth factor receptor recognize the nuclear epidermal growth factor receptor and (d) the nuclear receptor has an affinity for epidermal growth factor comparable to that of the plasma membrane receptor, but fewer (approximately 10%) nuclear receptors are available per protein unit compared with the plasma membrane.

In skin wounds, the chemokine CXCR3 receptor appears to play a key role in coordinating the switch from regeneration of the ontogenically distinct mesenchymal and epithelial compartments toward maturation. However, because CXCR3 equivalently binds four different ELR-devoid CXC chemokines (ie, PF4/CXCL4, IP-10/CXCL10, MIG/CXCL9, and IP-9/CXCL11), we sought to identify the ligand that coordinates epidermal coverage with the maturation of the underlying superficial dermis. Because CXCL11 (IP-9 or I-TAC) is produced by redifferentiating keratinocytes late in the regenerative phase when re-epithelialization is completed and matrix maturation ensues, we generated mice in which an antisense construct (IP-9AS) eliminated IP-9 expression during the wound-healing process. Both full and partial thickness excisional wounds were created and analyzed histologically throughout a 2-month period. Wound healing was impaired in the IP-9AS mice, with a hypercellular and immature dermis noted even after 60 days. Re-epithelialization was delayed with a deficient delineating basement membrane persisting in mice expressing the IP-9AS construct. Provisional matrix components persisted in the dermis, and the mature basement membrane components laminin V and collagen IV were severely diminished. Interestingly, the inflammatory response was not diminished despite IP-9/I-TAC being chemotactic for such cells. We conclude that IP-9 is a key ligand in the CXCR3 signaling system for wound repair, promoting re-epithelialization and modulating the maturation of the superficial dermis.

Epidermal growth factor (EGF) receptor-mediated cell migration plays a vital role in invasion of many tumor types. EGF receptor ligands increase invasiveness in vivo, but it remains unclear how consequent effects on intrinsic cell motility behavior versus effects on extrinsic matrix properties integrate to result in net increase of translational speed and/or directional persistence of migration in a 3D environment. Understanding this convolution is important for therapeutic targeting of tumor invasion, as key regulatory pathways for intrinsic versus extrinsic effects may not be coincident. Accordingly, we have undertaken a quantitative single-cell imaging study of glioblastoma cell movement in 3D matrices and on 2D substrata across a range of collagen densities with systematic variation of protease-mediated matrix degradation. In 3D, EGF induced a mild increase in cell speed and a strong increase in directional persistence, the latter depending heavily on matrix density and EGF-stimulated protease activity. In contrast, in 2D, EGF induced a similarly mild increase in speed but conversely a decrease in directional persistence (both independent of protease activity). Thus, the EGF-enhanced 3D tumor cell migration results only partially from cell-intrinsic effects, with override of cell-intrinsic persistence decrease by protease-mediated cell-extrinsic reduction of matrix steric hindrance.

Purpose: Mortality from head and neck squamous cell carcinoma (HNSCC) is usually associated with locoregional invasion of the tumor into vital organs, including the airway. Understanding the signaling mechanisms that abrogate HNSCC invasion may reveal novel therapeutic targets for intervention. The purpose of this study was to investigate the efficacy of combined inhibition of c-Src and PLCγ-1 in the abrogation of HNSCC invasion. Experimental Design: PLCγ-1 and c-Src inhibition was achieved by a combination of small molecule inhibitors and dominant negative approaches. The effect of inhibition of PLCγ-1 and c-Src on invasion of HNSCC cells was assessed in an in vitro Matrigel-coated transwell invasion assay. In addition, the immunoprecipitation reactions and in silico database mining was used to examine the interactions between PLCγ-1 and c-Src. Results: Here, we show that inhibition of PLCγ-1 or c-Src with the PLC inhibitor U73122 or the Src family inhibitor AZD0530 or using dominant-negative constructs attenuated epidermal growth factor (EGF)–stimulated HNSCC invasion. Furthermore, EGF stimulation increased the association between PLCγ-1 and c-Src in HNSCC cells. Combined inhibition of PLCγ-1 and c-Src resulted in further attenuation of HNSCC cell invasion in vitro. Conclusions: These cumulative results suggest that PLCγ-1 and c-Src activation contribute to HNSCC invasion downstream of EGF receptor and that targeting these pathways may be a novel strategy to prevent tumor invasion in HNSCC.

The fibroplasia noted during wound repair is resolved by fibroblast cell death. How fibroblasts undergo death and how this is prevented by trophic growth factors present during the regenerative phase are unknown at the molecular level. We examined a model of staurosporine-induced apoptosis in fibroblasts. We demonstrated that epidermal growth factor (EGF) stimulation of fibroblast NR6WT expressing human EGF receptors blocks staurosporine-induced apoptosis by inhibiting the activation of caspase-3. The survival effect of EGF on rescuing apoptotic NR6WT involves signaling pathways that derive from PI3K and Rac; the blockade of apoptosis is abolished when PI3K and Rac signals are inhibited simultaneously. Furthermore, by using KP372-1, a specific Akt inhibitor, we found that downstream of Akt signaling pathways is absolutely required for the EGF rescue from staurosporine-induced apoptosis in NR6WT. Interestingly, EGF prevention of apoptosis induced by tumor necrosis factor-alpha in the face of cycloheximide blockade of protein translation occurs via a different set of pathways as the simultaneous inhibition of extracellular signal-regulated kinase, Rac, and PI3K signaling did not eliminate EGF from rescuing fibroblasts in the face of this cytokine. These findings indicate that EGF receptor activation provides survival response against staurosporine-induced apoptosis through signal pathways of PI3K and Rac, which then may prevent the activation of caspase-3.

Background: Computational models of cell signaling networks typically are aimed at capturing dynamics of molecular components to derive quantitative insights from prior experimental data, and to make predictions concerning altered dynamics under different conditions. However, signaling network models have rarely been used to predict how cell phenotypic behaviors result from the integrated operation of these networks. We recently developed a decision tree model for how EGF-induced fibroblast cell motility across two-dimensional fibronectin-coated surfaces depends on the integrated activation status of five key signaling nodes, including a proximal regulator of transcellular contractile force generation, MLC (myosin light chain) [Hautaniemi et al, Bioinformatics 21: 2027 {2005}], but we have not previously attempted predictions of new experimental effects from this model. Results: In this new work, we construct an improved decision tree model for the combined influence of EGF and fibronectin on fibroblast cell migration based on a wider spectrum of experimental protein signaling and cell motility measurements, and directly test a significant and non-intuitive a priori prediction for the outcome of a targeted molecular intervention into the signaling network: that partially reducing activation of MLC would increase cell motility on moderately adhesive surfaces. This prediction was indeed confirmed experimentally: partial inhibition of the activating MLC kinase (MLCK) upstream using the pharmacologic agent ML-7 resulted in increased motility of NR6 fibroblasts. We further extended this exciting finding by showing that partial reduction of MLC activation similarly enhanced the transmigration of the human breast carcinoma cell line MDA-213 through a Matrigel barrier. Conclusion: These findings specifically highlight a central regulatory role for transcellular contractility in governing cell motility, while at the same time demonstrating the value of a decision tree approach to a systems "signal-response" model in discerning non-intuitive behavior arising from integrated operation a cell signaling network., National Institute of General Medical Sciences (U.S.) (Cell Migration Consortium grant U54-GM64346), National Institute of General Medical Sciences (U.S.) (grant R01-GM69668), United States. National Cancer Institute.Integrative Cancer Biology Program (grant U54-CA112967), Biocentrum Helsinki

MSC can act as a pluripotent source of reparative cells during injury and therefore have great potential in regenerative medicine and tissue engineering. However, the response of MSC to many growth factors and cytokines is unknown. Many envisioned applications of MSC, such as treating large defects in bone, involve in vivo implantation of MSC attached to a scaffold, a process that creates an acute inflammatory environment that may be hostile to MSC survival. Here, we investigated cellular responses of MSC on a biomaterial surface covalently modified with epidermal growth factor (EGF). We found that surface-tethered EGF promotes both cell spreading and survival more strongly than saturating concentrations of soluble EGF. By sustaining mitogen-activated protein kinase kinase-extracellular-regulated kinase signaling, tethered EGF increases the contact of MSC with an otherwise moderately adhesive synthetic polymer and confers resistance to cell death induced by the proinflammatory cytokine, Fas ligand. We concluded that tethered EGF may offer a protective advantage to MSC in vivo during acute inflammatory reactions to tissue engineering scaffolds. The tethered EGF-modified polymers described here could be used together with structural materials to construct MSC scaffolds for the treatment of hard-tissue lesions, such as large bony defects.Disclosure of potential conflicts of interest is found at the end of this article.

Metastatic seeding leads to most of the morbidity from carcinomas. However, little is known of this key event as current methods to study the cellular behaviors utilize nonrepresentative in vitro models or follow indirect subsequent developments in vivo. Therefore, we developed a system to visualize over a multiday to multiweek period the interactions between tumor cells and target organ parenchyma. We employ an ex vivo microscale perfusion culture system that provides a tissue-relevant environment to assess metastatic seeding behavior. The bioreactor recreates many features of the fluid flow, scale, and biological functionality of a hepatic parenchyma, a common site of metastatic spread for a wide range of carcinomas. As a test of this model, prostate and breast carcinoma cells were introduced. Tumor cell invasion and expansion could be observed by two-photon microscopy of red fluorescent protein (RFP)- and CellTracker-labeled carcinoma cells against a green fluorescent protein (GFP)-labeled hepatic tissue bed over a 14-day period. Tumors visible to the naked eye could be formed by day 25, without evident necrosis in the >0.3-mm tumor mass. These tumor cells failed to grow in the absence of the supporting three-dimensional (3D) hepatic microtissue, suggesting paracrine or stromal support function for the liver structure in tumor progression. Initial ultrastructural studies suggest that early during the tumor-parenchyma interactions, there are extensive interactions between and accommodations of the cancer and host cells, suggesting that the tumor-related epithelial-mesenchymal transition (EMT) reverts, at least transiently, to promote metastatic seeding. In sum, our 3D ex vivo organotypic liver tissue system presents a critical vehicle to examine tumor-host interactions during cancer metastasis and/or invasion. It also circumvents current limitations in assays to assess early events in metastasis, and provides new approaches to study molecular events during tumor progression.

Prostate cancer (PCa) is the leading cause of cancer and the second leading cause of cancer death among men in the US, with tumor metastasis being the main reason behind high rates of mortality and morbidity. How these tumors escape the molecular signals that prevent epithelial cell migration from the prostate is being deciphered. A key motility ‘stop’ signaling network is via chemokines that bind to the common receptor CXCR3. CXCR3 is a G protein coupled receptor whose splicing is differentially regulated in prostate cancer. We have found over-expression of the CXCR3A splice isoform in prostate cancers and cell lines whereas normal prostate epithelium expresses by and large only the CXCR3B isoform, representing a possible switch in the expression between the CXCR3 splice variants. As these variants link differentially to pro- and anti-invasion signaling pathways, this may explain the relentless progression of the prostate carcinoma cells. The switch is present at both the RNA and protein level which suggests that the altering factor is at the transcriptional control level. In a series of PCa lines, the CXCR3 gene was scanned for insertions; deletions or substitutions that introduce a splice silencer; enhancer or a premature stop codon. Surprisingly, the gene was free from mutations except for a polymorphism in the intron in one of the cancer cell lines. A mutation is a permanent change whereas an epigenetic change is a reversible change that confers the cell with the ability to wield control over the expression of the isoforms at a given time. This suggests presence of a possible epigenetic change and explains the absence of mutations. To confirm the aforementioned, the CXCR3 epigenome was analyzed to find the intronic region differentially methylated at four different nucleotides while the upstream putative promoter was not. Strikingly, two of the differentially methylated sites were flanking splice sites while two of were adjoining the NK-2 transcription repressor binding site. A bichromatic CXCR3 reporter minigene has been designed to study the cell type specific expression pattern of the variants and the effect of each differentially methylated CpG nucleotide on splicing. Upon transfection of the wildtype minigene, it was found that the cancer cells express both the isoforms and as expected higher levels of CXCR3A when compared to normal prostate epithelial cells. The switch of isoforms was observed as: one subpopulation of cells that express both the isoforms; another that express solely one of the isoforms: either CXCR3A or CXCR3B. The mutant with guanines at the differentially methylated sites would validate the epigenomic link to splicing. The use of this reporter will be presented to dissect the plasticity of this epigenetic change during dissemination in experimental models of prostate cancer. Citation Format: Devi Sharanya Sampath Kumar, Alan Wells. CXCR3 epigenome switches splice variants in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2973. doi:10.1158/1538-7445.AM2013-2973

The Luminex-based class I and II human leukocyte antigen (HLA) antibody testing is widely used for organ transplantation. However, false negative results can occur due to prozone inhibition when complement binds to antigen-bound HLA antibodies, thereby blocking access of secondary antibodies. The addition of ethylenediaminetetraacetic acid (EDTA) to serum can decrease complement-mediated prozone effect by chelating calcium needed for C1q assembly. The aim of this …

Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) spectra were examined for any unique spectral features that could expedite the clinical microbiology laboratory’s identification of Klebsiella pneumoniae carbapenemase (KPC)-producing Escherichia coli (KPC-EC). Previously at our institution, the plasmids carrying bla KPC were investigated in 20 KPC-EC isolates, and various plasmids including IncN (4/20), ColE (2/20), and others were …

Keloid scars after skin trauma are a significant clinical problem, especially in black populations, in which the incidence of keloids has been estimated at 4-16%. Keloids are abnormal dermal proliferative scars secondary to dysregulated wound healing. Despite several biochemical studies on the role of extracellular matrix proteins and growth factors during keloid formation, we still do not know what molecules and signals induce this change. Fibroblasts are thought to be the major inductive cell for keloid scar formation. The aim of this study was to identify gene expression patterns that characterize keloid fibroblasts; identifying such genetic disequilibrium may shed light on the molecular signaling events responsible for keloid formation. In this study, we performed gene expression analysis of fibroblasts isolated from keloid lesions from three individuals in comparison with the fibroblasts isolated from normal skin using the Affymetrix U133a chip (22,284 genes and expression sequence tags). We found through J5 test score expression analysis that among 22,284 genes, there were 43 genes that were overexpressed and five genes were underexpressed in keloid fibroblasts when compared with dermal fibroblasts from persons without keloids. The overexpression of three genes not previously reported as being up-regulated in keloids (annexin A2, Transgelin, and RPS18) was confirmed by real-time polymerase chain reaction. Certain overexpressed genes were similar to previous biochemical observations on the protein levels of these overexpressed genes during keloid formation. We also report for the first time that a few tumor-related genes are overexpressed in keloid fibroblasts.

The mortality and morbidity from prostate carcinomas result primarily from their progression to invasive and metastatic state. Recent studies have uncovered the crucial role of cell motility in dictating these highly orchestrated biological events. This review will focus on the intracellular and extracellular factors that govern cell motility during prostate tumor cell invasion. The role of receptor signaling systems, including those for epidermal growth factor and hepatocyte growth factor, chemokine receptors, and sex steroid hormones (estrogen and testosterone) will be discussed with reference to their modulation of cellular invasive potential by regulating motility. Lastly, detailed cellular biophysical events and the underlying biochemical molecules that govern these distinct events will be discussed with the goal that targeting these individual events using the emerging genetic and pharmacological interventions can lead to more specific and successful therapies against prostate cancer.

Growth factor-induced migration is a rate-limiting step in tumour invasiveness. The molecules that regulate this cellular behaviour would represent novel targets for limiting tumour cell progression. Epidermal growth factor (EGF) receptor (EGFR)-mediated motility, present in both autocrine and paracrine modes in prostate carcinomas, requires de novo transcription to persist over times greater than a few hours. Therefore, we sought to define specific signalling pathways that directly alter cellular transcription. Signal transducer and activator of transcription 3 (STAT3) is activated, as determined by electrophoretic motility shift assays, by EGFR in DU145 and PC3 human prostate carcinoma cells in addition to the motility model NR6 fibroblast cell line. Inhibition of STAT3 activity by antisense or siRNA downregulation or expression of a dominant-negative construct limited cell motility as determined by an in vitro wound healing assay and invasiveness through a extracellular matrix barrier. The expression of constitutively activated STAT3 did not increase the migration, which indicates that STAT3 is necessary but not sufficient for EGFR-mediated migration. These findings suggest that STAT3 signalling may be a new target for limiting prostate tumour cell invasion. In a microarray gene analysis of what transcription units are altered by EGF in a STAT3-dependent manner we found that the expression of motility-limiting VASP protein and the apoptosis nexus caspase 3 were both downregulated upon EGF exposure. These findings suggest a molecular basis for the STAT3 dependence of EGFR-mediated prostate tumour progression.

Tumor progression to the invasive phenotype occurs secondary to upregulated signaling from growth factor receptors that drive key cellular responses like proliferation, migration, and invasion. We hypothesized that Protein kinase Cdelta (PKCdelta)-mediated transcellular contractility is required for migration and invasion of prostate tumor cells. Two invasive human prostate cancer cell lines, DU145 cells overexpressing wildtype human EGFR (DU145WT) and PC3 cells, were studied. PKCdelta is overexpressed in these cells relative to normal prostate epithelial cells, and is activated downstream of EGFR leading to cell motility via modulation of myosin light chain activity. Abrogation of PKCdelta using Rottlerin and specific siRNA significantly decreased migration and invasion of both cell lines in vitro. Both PKCdelta and phosphorylated PKCdelta protein levels were higher in human prostate cancer tissue relative to normal donor prostate as assessed by Western blotting and immunohistochemistry. Thus, we conclude that PKCdelta inhibition can limit migration and invasion of prostate cancer cells.

Angiogenesis plays a critical role in wound repair. Endothelial cells present CXC receptor 3 (CXCR3) for chemokines expressed late in wound regeneration. To understand the physiological role CXCR3 plays in regulating endothelial function, we analyzed the ability of a CXCR3 ligand, IP-10 (CXCL10), to influence endothelial cell tube formation. Treatment of endothelial cells with IP-10 in the presence of vascular endothelial growth factor (VEGF) inhibited tube formation on growth factor-reduced Matrigel and in a subcutaneous Matrigel plug. Furthermore, IP-10 significantly inhibited VEGF-induced endothelial motility, a response critical for angiogenesis. Previous work showed that CXCR3 ligandation initiates protein kinase A (PKA) phosphorylation-dependent inhibition of m-calpain, required for induced cell motility, in fibroblasts but not epithelial cells. Here we show that CXCR3 activation in endothelial cells induces an increase in cAMP and PKA activation. Treatment of endothelial cells with Rp-8-Br-cAMP, an inhibitor of PKA, or small interference RNA to PKA was able to reverse the inhibitory effects of IP-10 on VEGF-mediated tube formation and motility. Importantly, treatment of endothelial cells with VEGF induced the activation of m-calpain, but costimulation with IP-10 significantly decreased this activity. Using Rp-8-Br-cAMP, we show blocking PKA reversed the IP-10 inhibition of VEGF-induced m-calpain activity. These data indicate that the activation of CXCR3 inhibits endothelial tube formation through a PKA mediated inhibition of m-calpain. This provides a means by which late wound repair signals limit the angiogenesis driven early in the wound response process.

Nearly half of breast cancer metastases will become clinically evident 5 or more years after the cancer was seemingly ablated. This implies that metastatic cancer cells survived over this extended timeframe without emerging as detectable nodules. Breast cancer metastatic dormancy has yet to be universally defined, and is not well understood as a potential means by which clinically evident metastases emerge after being undetected for years (or decades) from the primary tumor diagnosis. Various mechanistic and microenvironmental factors may work independently, collaboratively (or both) in order to allow a dormant metastatic breast cancer cell (or small cluster) to survive long term. At least one tumor dormancy hallmark is likely required: that of E-cadherin re-expression in order to form heterotypic connections between the carcinoma cells and the liver parenchyma. This re-expression is known as the mesenchymal-to-epithelial-reverting-transition (MErT). This re-expression provides not only survival signals to the breast cancer cells but may also establish cell polarity and protection from autocrine signaling (or extracellular factors) that otherwise may prompt the breast cancer cells to initiate metastatic outgrowth. The purpose of this study was to determine whether non-parenchymal liver cells (NPC) contribute to metastatic competency, initiate mesenchymal to epithelial reverting transition (MErT), and trigger metastatic dormancy. We co-cultured human or rat hepatic isolations with highly metastatic human immortalized breast cancer cells (MDA-MB-231). The hepatocytes, non-parenchymal hepatic supernatant, and liver sinusoidal endothelial cell enriched populations were independently cultured with the breast cancer cells in standard monolayer cultures. Because MDA-MB-231 cells do not survive in the hepatocyte culture media alone, we were able to assess the unique properties imparted by the experimental cultures. Confocal immunofluorescent imaging for proliferation and epithelial markers revealed a mesenchymal phenotypic transition only in the hepatocyte/breast cancer cultures. Interestingly the NPC/breast cancer co-cultures promoted cancer cell proliferation (but lower than with hepatocytes included) but did not impart carcinoma epithelial reversion. The liver sinusoidal endothelial cell (LSEC)-enriched co-cultures exhibited much lower breast cancer cell proliferation hinting toward a possible dormancy, though E-cadherin reversion was not detected. These data suggest that perturbations of the parenchymal and non-parenchymal cell ratios in the liver metastatic microenvironment may contribute to metastatic dormancy initiation or stability. Citation Format: Donald P. Taylor, Alan Wells. Liver nonparenchymal cells drive metastatic breast cancer survival but fail to initiate mesenchymal to epithelial reversion. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C93.

Bone marrow mesenchymal stem cells (BMMSCs) are pluripotent cells capable of differentiating into several cell types and are thus an attractive cell source for connective tissue engineering. A challenge in such a use is expansion and directed seeding in vitro, requiring proliferation and survival, and directed migration, respectively, prior to functional differentiation. The epidermal growth factor (EGF) receptor (EGFR) is the prototypal growth factor receptor and elicits these responses from a wide variety of stromal, epithelial, and endothelial cells. Ligands for this receptor are appealing for use in tissue engineering because they are relatively resistant to biological extremes and amenable to high-volume production. Therefore, we determined whether an EGFR ligand, EGF, could be used for ex vivo expansion of BMMSCs. EGF stimulated motility in rat and immortalized human BMMSCs. EGF-induced proliferation was observed in immortalized human BMMSCs but was not apparent in rat BMMSCs under our experimental conditions. EGF did not, however, rescue either type of BMMSC from apoptosis due to lack of serum. During our examination of key signaling intermediaries, EGF caused robust phosphorylation of extracellular signal-regulated protein kinase (ERK) and protein kinase B/akt (AKT) but only minimal phosphorylation of EGFR and phospholipase C-γ in rat BMMSCs, whereas in the human BMMSCs these intermediaries were all strongly activated. EGF also induced robust ERK activation in primary porcine mesenchymal stem cells. EGF pretreatment or cotreatment did not interfere with secondarily induced differentiation of either type of BMMSC into adipogenic or osteogenic lineages. Platelet-derived growth factor (PDGF) effects were similar to but not additive with those elicited by EGF, with some quantitative differences; however, PDGF did interfere with the differentiation of these BMMSCs. These findings suggest that EGFR ligands could be used for ex vivo expansion and direction of BMMSCs.

Chemokine receptor 7 (CCR7) upregulation, which mediates immune cell survival and migration to lymph nodes, has recently been associated with nodal metastasis of squamous cell carcinoma of the head and neck (SCCHN). However, the mechanism of CCR7 in tumor progression, its downstream signaling mediators, and interactions with other pathways contributing to metastasis of SCCHN have not been determined. We hypothesized that inflammatory chemokine-mediated signals could also promote tumor proliferation and mitogenic effects. Functional assays showed that chemotaxis and invasion of metastatic SCCHN cells were dependent on phosphoinositide-3 kinase (PI3K) and its substrate, activated phospholipase Cgamma-1. In addition, treatment of CCR7(+) metastatic SCCHN cells with CCL19 (MIP-3beta) showed rapid activation of the prosurvival, PI3K/Akt pathway. Transactivation of EGFR-mediated and mitogen-activated protein kinase signaling pathways, which can promote migration and survival in parallel, did not appear to contribute to the functional or biochemical effects of CCR7 stimulation. Thus, proinflammatory chemokine signals that mediate activation, trafficking and survival of tumor-infiltrating immune cells in the tumor microenvironment actually appear to induce signals for progression of cancer cells. The CCR7-mediated pathway in metastatic SCCHN cells functions independently of EGFR signal transduction and therefore may represent an additional target for therapeutic intervention to prevent tumor progression and metastasis.

Originally characterized as a growth factor for erythrocytes, erythropoietin (EPO) is used to treat anemia and fatigue in cancer patients receiving radiation therapy and chemotherapy. EPO and the EPO receptor (EPOR) are expressed in nonhematopoietic cells and cancers. However, the role of EPO and EPOR within nonhematopoietic cancer cells remains incompletely understood. Although a recent clinical trial demonstrated worse tumor control and survival in head and neck cancer patients treated with EPO, the role of EPO and EPOR in head and neck squamous cell carcinoma (HNSCC) has not been examined. In the present study, we demonstrate the previously unrecognized EPO-mediated invasion by HNSCC cells through the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway. Furthermore, we confirmed the expression of EPO and EPOR in a panel of human HNSCC cell lines and tissue specimens. Pharmacological doses of EPO also had a limited proliferation effect in these cell lines. These results define a novel role for EPO in mediating tumor cell invasion. Increased levels of EPO and EPOR in lymph node metastases as compared to primary tumors from HNSCC patients further support the role of EPO/EPOR in HNSCC disease progression and metastasis.

Our overall objective is to understand which tumor cell behaviors contribute to invasion and metastasis. This would allow rationale approaches to limit these aspects of tumor progression. While great strides have defined critical molecular determinants, the current experimental models of tumor invasion limit the dissection of complex cellular responses. In vitro assays do not capture tumor/host relations or relevant tissue architecture and physiology. In vivo model systems provide the relevant organism contexts but cannot readily be manipulated. Quantal advances would be enabled by combining the best attributes direct manipulation of tumor and host, long-term visualization, and tissue relevant architecture. Our central premise is that an ex vivo organotypic liver tissue system can provide an environment to study tumor cell invasion and metastasis. Our objective is to utilize a physiologically relevant microreactor that has proved suitable for organotypic liver culture to investigate metastatic seeding. The sub-millimeter scale of this liver allows for real-time imaging over weeks in culture. We established this system to determine what step is rate-limiting for tumor progression. We have now established an organotypic liver tissue culture that supports metastatic establishment and growth. This will be used to probe the molecular steps that are key to this progression.

Regulated cell motility is critical to wound healing (25, 35). During repair, tissue deposition and remodeling by the immigrant fibroblasts and keratinocytes result in the regeneration of an intact skin barrier and functional organ. The cells from the remaining epidermal and dermal layers must proliferate and migrate to repopulate the nascent wound. The basal keratinocytes undergo a transition that enables such repopulation while the provisional matrix is invaded by fibroblasts as the first step in regenerating the future dermal layer. The numerous growth factors present throughout repair, including high levels of epidermal growth factor (EGF) receptor (EGFR) ligands such as HB-EGF and transforming growth factor alpha, are thought to promote these mitogenic and motogenic responses (26, 35, 45, 50, 60). However, the process of cell repopulation is limited late in the process of healing to prevent fibroplasia and excess matrix deposition. Late in the repair process, members of the Alu-Leu-Arg (ELR)-negative family of CXC chemokines appear (13, 42). It has been proposed that the migration of fibroblasts and keratinocytes is controlled by the waves of these growth factors and chemokines produced throughout wound repair (9, 35, 42, 44, 60). This would include signals to promote as well as inhibit cell migration. Active cell locomotion requires the coordination of a number of cellular processes that should be common among cell types (18, 28). Thus, as numerous external signals can modulate cell motility, two key questions are which biochemical pathway is actuated to promote migration during regeneration and whether these differ between cell types. Any such signaling pathway needs to affect key biophysical processes. During cell migration, tail de-adhesion may be rate-limiting; in experimental models, failure to detach limits cell motility (23, 44). Activation of calpain (EC 3.4.22.17), an intracellular limited protease, is required for integrin-mediated tail de-adhesion on moderately and highly adhesive substrata (30, 37) and for growth factor-induced motility (17, 43). This intracellular protease is a key switch, as calpain inhibitors convert EGFR-mediated signals from cell motility to matrix contractility (1). Thus, calpain activators appear to shift a wide range of cells to motility-permissive adhesion regimens, while inhibitory signals for calpain block productive locomotion. As such, we proposed that keratinocyte motility was dependent on calpain activity. This requirement for calpain activity provides a target for regulating cell motility (18). Confounding any analysis, two calpain isoforms with seemingly identical target specificities are present in practically all cells (48). In vitro, calpain 1 (μ-calpain) is activated at nearly micromolar concentrations of calcium; calpain 2 (M-calpain) requires millimolar calcium levels. While calcium fluxes have been postulated to regulate μ-calpain, this has yet to be demonstrated conclusively in living cells (5), and the signaling cascade that triggers this isoform during cell locomotion remain undefined (30). Furthermore, the physiologically relevant activators of M-calpain are unknown since intracellular calcium levels fail to reach the nearly millimolar concentrations required in vitro (22). Still, we know that plasma membrane-localized M-calpain is activated subsequent to growth factor signaling by direct extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAP kinase) phosphorylation (12, 16, 17, 44). However, what signals and respective intracellular signaling pathways operate during reepithelialization by keratinocytes remains an open question (42). In an initial exploration (43, 44), we reported that ELR-negative CXC chemokines, present during the process of wound repair (13, 42), can block fibroblast motility by preventing activation of M-calpain and subsequent de-adhesion, demonstrating that this might be a physiologically operative pathway. Of this family of chemokines, interferon-inducible protein 10 (IP-10) appears to be produced by the neovasculature deep in the dermis (13). A related ELR-negative CXC chemokine, IP-9, also called beta-R1 (40), H174 (24), and I-TAC (10), is produced by basal keratinocytes in response to immune-mediated injuries (52). Previously, we demonstrated that IP-9 is a wound response factor (42). These ELR-negative CXC chemokines were originally found as modulators of cells of the hematopoietic lineage, but chemokine receptors have been found on endothelial and epithelial cells (36, 38, 44, 47, 63). The ELR-negative members of the CXC family of chemokines, all of which bind to a common CXCR3 receptor (2, 14, 27), inhibit endothelial cell proliferation, migration (21, 31, 51), and fibroblast migration (44). These appear to act dominantly over promitogenic and promotility chemokines and growth factors (43, 44). IP-9 is a wound response factor situated in the right place to limit fibroblast repopulation and promote the remodeling phase (42). However, with IP-9 being produced by keratinocytes in or near the wound bed, the situation for the reepithelializing keratinocytes is of great interest; would this chemokine, which serves to “mature” the dermis, also have the collateral effect of slowing reepithelialization? Here we report that keratinocyte motility is promoted by both EGF and IP-9 through their activation of calpains. Furthermore, IP-9 does not block EGF-induced motility in keratinocytes, opposite to its effects on fibroblasts. Keratinocyte motility induced by EGF requires the pathway previously described in fibroblasts, which culminates in activation of the calpain 2 isoform, M-calpain (17). In undifferentiated keratinocytes, IP-9 also activated calpain, though it was the μ-calpain isoform and not the M-calpain isoform that was both triggered and required. This chemokine activation occurred through a phospholipase Cβ (PLCβ)-mediated calcium flux in distinction to the ERK MAP kinase signaling cascade that growth factors utilize to activate M-calpain (17). These two pathways converge at cell-diminished cell adhesion to substratum as mirrored in vinculin aggregate disassembly and cleavage of the focal adhesion kinase (FAK). This is the first demonstration, to our knowledge, of different isoforms of calpain being activated by distinct signals in the same cell to accomplish the same phenotypic end point, cell migration. We also show for the first time that IP-9 increased the intracellular calcium flux and resulted in triggering of the μ-calpain isoform, in turn resulting in productive cell motility.

Motivation: Signal transduction cascades governing cell functional responses to stimulatory cues play crucial roles in cell regulatory systems and represent promising therapeutic targets for complex human diseases. However, mathematical analysis of how cell responses are governed by signaling activities is challenging due to their multivariate and non-linear nature. Diverse computational methods are potentially available, but most are ineffective for protein-level data that is limited in extent and replication. Results: We apply a decision tree approach to analyze the relationship of cell functional response to signaling activity across a spectrum of stimulatory cues. As a specific example, we studied five intracellular signals influencing fibroblast migration under eight conditions: four substratum fibronectin levels and presence versus absence of epidermal growth factor. We propose techniques for preprocessing and extending the experimental measurement set via interpolative modeling in order to gain statistical reliability. For this specific case study, our approach has 70% overall classification accuracy and the decision tree model reveals insights concerning the combined roles of the various signaling activities in governing cell migration speed. We conclude that decision tree methodology may facilitate elucidation of signal--response cascade relationships and generate experimentally testable predictions, which can be used as directions for future experiments. Contact: [email protected]

Cell adhesion and its role during cell spreading and motility are central to normal development and homeostasis, including its effects on immune response and wound repair and tissue regeneration. Disruption of cell adhesion impacts not only the healing process but promotes tumor invasion and metastasis. A family of intracellular, limited proteases, the calpains, has recently been shown to be a key molecular control point in attachment of cells to the surrounding matrix. Herein, the two main and ubiquitously expressed calpain isoforms will be introduced as to their modes of regulation and the current status of research will be discussed as to how these calpains might function in the biophysical process of adhesion and biological cellular responses of spreading and motility.

HER2, a member of the epidermal growth factor receptor (EGFR) tyrosine kinase family, functions as an accessory EGFR signaling component and alters EGFR trafficking by heterodimerization. HER2 overexpression leads to aberrant cell behavior including enhanced proliferation and motility. Here we applied a combination of computational modeling and quantitative experimental studies of the dynamic interactions between EGFR and HER2 and their downstream activation of ERK to understand this complex signaling system. Using cells expressing different levels of HER2 relative to the EGFR, we could separate relative contributions of EGFR and HER2 to signaling amplitude and duration. Based on our model calculations, we demonstrated that, in contrast with previous suggestions in the literature, the intrinsic capabilities of EGFR and HER2 to activate ERK were quantitatively equivalent. We found that HER2-mediated effects on EGFR dimerization and trafficking were sufficient to explain the observed HER2-mediated amplification of epidermal growth factor-induced ERK signaling. Our model suggests that transient amplification of ERK activity by HER2 arises predominantly from the 2-to-1 stoichiometry of receptor kinase to bound ligand in EGFR/HER2 heterodimers compared with the 1-to-1 stoichiometry of the EGFR homodimer, but alterations in receptor trafficking yielding increased EGFR sparing cause the sustained HER2-mediated enhancement of ERK signaling.

Cell migration along the correct paths is critical to normal organogenesis and tissue repair, and goes awry in tumor dissemination. Understanding the molecular cues that control such migration would provide opportunities to control these events. Recent findings in Drosophila highlight the role of the epidermal growth factor receptor (EGFR) in chemotaxis and pathfinding during development. These studies, and others performed in vertebrates, support a central role for EGFR-mediated chemotaxis. However, the signal or signals that then enable cells to further migrate independently of this chemotactic effect are not known. Three possible scenarios are discussed: that EGFR signaling sensitizes cells to new cues, that EGFR signaling is altered so that the cell responds predominantly to other motility signals that are now "unblinded," and that EGFR signaling still provides the motile signal but in a chemokinetic manner with matrix components providing the directionality. There are precedents for all three scenarios, but which one proves true will define the window of opportunity for regulating cell migration.

How m-calpain is activated in cells has challenged investigators because in vitro activation requires near-millimolar calcium. Previously, we demonstrated that m-calpain activation by growth factors requires extracellular signal-regulated kinase (ERK); this enables tail deadhesion and allows productive motility. We now show that ERK directly phosphorylates and activates m-calpain both in vitro and in vivo. We identified serine 50 as required for epidermal growth factor (EGF)-induced calpain activation in vitro and in vivo. Replacing the serine with alanine limits activation by EGF and subsequent cell deadhesion and motility. A construct with the serine converted to glutamic acid displays constitutive activity in vivo; expression of an estrogen receptor fusion construct produces a tamoxifen-sensitive enzyme. Interestingly, EGF-induced m-calpain activation occurs in the absence of increased intracellular calcium levels; EGF triggers calpain even in the presence of intracellular calcium chelators and in calcium-free media. These data provide evidence that m-calpain can be activated through the ERK cascade via direct phosphorylation and that this activation may occur in the absence of cytosolic calcium fluxes.

Myopodin was previously reported as a gene that was frequently deleted in prostate cancer. This gene shares significant homology with a cell shape-regulating gene, synaptopodin. Myopodin was shown to bind actin and to induce actin bundling when cells were stimulated. To clarify the functional role of myopodin in prostate cancer, several assays were performed to evaluate the tumor suppression activity of myopodin. Our results indicate that myopodin inhibits tumor growth and invasion both in vitro and in vivo. The activity of tumor suppression of myopodin is located at the C-terminus region. To further evaluate the role of myopodin in suppressing the invasiveness of prostate cancer, an expression analysis of myopodin protein was performed in prostate tissues. The results indicate that down-regulation of myopodin expression occurs mostly in invasive stages of prostate cancer, implying a potential invasion suppression role for myopodin in prostate cancer. In addition, hemizygous deletion and down-regulation of myopodin expression occur in three aggressive prostate cancer cell lines. All these results support the hypothesis that myopodin functions as a tumor suppressor gene to limit the growth and to inhibit the metastasis of cancer cells.

Tumor cell motility and invasion have been linked to upregulated signaling from both the epidermal growth factor receptor (EGFR) and that for urokinase-type plasminogen activator (uPAR). However, we do not know whether these events are interdependent or unrelated, despite the obvious diagnostic and therapeutic implications. Gene microarray analyses have suggested that EGFR signaling via phospholipase C-gamma (PLCgamma) induces uPAR transcription. We utilized two sublines of the DU145 human prostate carcinoma cell line that are genetically engineered to differentially activate the EGFR/PLCgamma cascade and are variously invasive in vitro and in vivo. uPAR protein levels in these cells were found to be dependent on PLC signaling, pharmacologic inhibition of PLC signaling reduced uPAR expression. To determine whether uPAR was a required element in EGFR-mediated invasion, we stably expressed uPAR cDNA in either sense or antisense orientation in the two DU145 sublines. Interestingly, uPA production was modulated in parallel, although to a lesser degree, with uPAR in these sublines. Antisense to uPAR significantly restricted invasion of the highly invasive DU145 WT cells through Matrigel and reduced aggressiveness of tumors in nude mice. Up-regulation of uPAR significantly increased the invasiveness of the moderately invasive DU145 parental (DU145 P) cells through Matrigel, but this increased invasiveness was not seen in mice. uPA activity appears to contribute to invasiveness at least through Matrigel, as antibody to uPA or amiloride limited the transmigration. These results support a model of tumor invasion promoted by autocrine EGFR signaling involving reinforcing altered gene expression, of uPAR at least, that further induces cell motility. Herein, a number of key molecules whose expression levels are interrelated, including both EGFR and uPAR, are required but none are sufficient in the absence of other keys molecules in promoting tumor progression.

As fibroblasts near senescence, their responsiveness to external signals diminishes. This well-documented phenomenon likely underlies physiological deterioration and limited tissue regeneration in aging individuals. Understanding the underlying molecular mechanisms would provide opportunities to ameliorate these situations. A key stimulus for human dermal fibroblasts are ligands for the epidermal growth factor receptor (EGFR). We have shown earlier that EGFR expression decreases by about half in near senescent fibroblasts (Shiraha et al., 2000, J. Biol. Chem. 275 (25), 19343-19351). However, as the cell responses are nearly absent near senescence, other aging-related signal attenuation changes must also occur. Herein, we show that EGFR signaling as determined by receptor autophosphorylation is diminished over 80%, with a corresponding decrease in the phosphorylation of the immediate postreceptor adaptor Shc. Interestingly, we found that this was due at least in part to increased dephosphorylation of EGFR. The global cell phosphotyrosine phosphatase activity increased some threefold in near senescent cells. An initial survey of EGFR-associated protein tyrosine phosphatases (PTPases) showed that SHP-1 (PTPIC, HCP, SHPTP-1) and PTPIB levels are increased in parallel in these cells. Concomitantly, we also discovered an increase in expression of receptor protein tyrosine phosphatase alpha (RPTPalpha). Last, inhibition of protein tyrosine phosphatases by sodium orthovanadate in near senescent cells resulted in increased EGFR phosphorylation. These data support a model in which, near senescence, dermal fibroblasts become resistant to EGFR-mediated stimuli by a combination of receptor downregulation and increased signal attenuation.

Wound healing is a complex process ofcell colonization, matrix deposition,and remodeling, requiring multiple cellfunctions to act in coordinated manner.Focusing on dermal tissue regenerationas a prominent example, a relativelysimplifiedviewcanbeproffered:properhealing requires fibroblast migrationinto the wound, synthesis and secretionof extracellular matrix components,and active contraction-based reorgan-ization of the matrix around the occu-pying cells. These actions occur in anoverlapping but sequenced order inthe face of a multitude of external cuesfrom a changing environment. Furthercomplicating this situation is the factthat these stages of wound repair occurasynchronously across a larger, healingwound. The ways by which thesediverse cell behaviors—locomotion,matrix production, and tissue remod-eling—interact to ultimately yield ei-therpropertissuephysiologyorvariouskinds of pathology remain poorly un-derstood,even to the issueof the natureof the cells involved in any or all ofthese functions (Tomasek et al., 2002).For investigators in the biophysicscommunity, an especially germanefacet of this complicated phenomenonis the relationship between cell loco-motion and matrix reorganization,particularly since the matrix reorgani-zation requires matrix contraction. Attheir core both the cellular immigrationand the matrix contraction arise fromintracellular cytoskeletal force genera-tion transmitted to the extracellularmatrix via highly regulated cell/matrixadhesion sites (Tamariz and Grinnell,2002). Controversy has existed in thewound healing and tissue morphogen-esis field about whether compaction ofextracellular matrix derives, on the onehand, from migratingcells asthey exerttraction during locomotion, or, on theother hand, by nonmigrating cells con-verting their intracellular contractileforce into traction. In fact, fibroblastmotility can be converted into matrixcompaction by inhibiting molecularmechanisms involved in regulated re-leaseofcell/matrixadhesionsoperatingin migration (Allen et al., 2002). Thus,it is exceedingly difficult to parsecontributions of underlying molecularmechanisms to overall wound healingand tissue morphogenesis in terms ofactive cell migration versus cell motil-ity-derivedmatrix deformations.It maysimply be that they are part-and-parcelaspects of the same underlying motilitymachinery modulated by external sig-nals that abrogate key events. It is,therefore, crucial to gain this capabilityfor analyzing the ‘‘multitasking’’ cellscan accomplish with their force gen-eration and transmission regulation inorder to test hypotheses that can lead toimproved molecular therapeutics forwound regeneration and particularlywound strengthening.A major advance toward this impor-tant goal is provided by the new workby Shreiber et al. in this issue of theBiophysical Journal (Shreiber et al.,2003), by simultaneous quantitativemeasurement of cell migration andmatrixdeformationforfibroblastswith-in three-dimensional collagen gels asfunctionsoftimeoveraperiodofhours.These gels are cylindrical andanchoredat their axial ends, so that matrix de-formationoccurs in the radial direction.The automated microscopy and imageanalysis technology needed for thiscapability had been demonstrated pre-viously by the Tranquillo laboratoryat Minnesota in recent years, but thecrucial extension here determination oftime-varying characteristics of thesecellbehavioralfunctionsconcomitantlyenabling analysis of dynamic changesin locomotion and remodeling reflect-ing progressions of the extracellularcontext (e.g., matrix composition andcompliance) as well as cellular pheno-typic properties (due to gene expres-sion modulation, for instance).In this new study, the Tranquillogroup compared dynamic cell migra-tion and matrix compaction propertiesof human foreskin fibroblasts (HFFs)and rat dermal fibroblasts (RDFs) inresponse to serum. Both cell typesgenerally showed increasing values ofthe random migration coefficient,l (analogous to a molecular diffusioncoefficient), as time progressed, whilethevaluesofthecelltractioncoefficent,s

Regulation of calpain by phosphorylation has often been suggested, but has proved difficult to detect. Calpains extracted from mammalian tissue are reported to contain 2–4 mol phosphate/mol of enzyme distributed over multiple sites, but phosphate groups are not detectable in the X-ray structures of recombinant calpain. Some serine and threonine residues in the large subunit of rat m-calpain were converted to aspartic or glutamic acid residues, at sites suggested by previous studies, to assess the probable effects of phosphate groups on the enzyme. Expression of the mutant calpains in Escherichia coli, and their heat stabilities, did not differ from those of the wild-type enzyme. m-Calpains with the mutations Ser50Asp, Ser50Glu, Ser67Glu, and Thr70Glu had the same specific activity and Ca2+ requirement as the wild-type enzyme. In contrast, Ser369Asp-, Ser369Glu-, and Thr370Glu-m-calpain were inactive. This result is consistent with the recent report that phosphorylation at position 369 or 370 in vivo reduced m-calpain activation.

Much is known about this ubiquitous family of limited intracellular proteases. Many investigators have defined the extended family and begun to establish structural bases of calpain activation and regulation. In addition, a number of functional roles have been established by calpain family selective inhibitors, which in turn have provided potential target proteins. However, much remains to be learned about this complex family of enzymes. Four decades of study have provided much understanding of the calpain family of intracellular cysteine proteases. Due to the limitations of investigative tools, earlier work focused on in vitro regulation and activities of these proteases, primarily the two ubiquitous isoforms μ- (calpain I) and m-calpain (calpain II). Structure-function studies have culminated in elucidating the molecular structure of the two ubiquitous calpains and deciphering how these enzymes might be activated and modulated. More recently, investigators have focused on connecting calpain function to physiology and pathology, particularly in regard to motility during wound repair, injury-mediated apoptosis in stroke and ischemia, protein degradation in muscular dystrophies, and susceptibility for non-insulin dependent diabetes mellitus.

Tumor invasion marks a critical point in cancer progression; it is a harbinger of morbidity and mortality. Thus, the cellular events that enable the invasive phenotype are under intense investigation. Epstein-Barr virus (EBV) is associated with a number of cancers, including Burkitt lymphoma (BL) and nasopharyngeal carcinoma (NPC) and is suspected to contribute to their tumorigenesis. On average, 8% of gastric carcinomas have been shown to carry this virus. To explore whether the presence of EBV in gastric carcinoma contributes to tumor progression in this predominantly invasive carcinoma, we examined a panel of 2 in vitro EBV-infected human gastric cancer cell line sublines and their mock-infected AGS parental control line. We found EBV infection caused a marked increase in transmigration of a Matrigel barrier (415% and 303%, p < 0.05, for the 2 infected lines). This correlated with increased motility of these sublines (233% and 140%, p < 0.05). As this pattern of increased motility leading to a more pronounced enhancement of invasion has been noted in other tumor cells, we explored the roles of autocrine signaling pathways previously implicated in carcinoma motility and invasion. Inhibitors to the epidermal growth factor receptor (EGFR) (PD153035), phospholipase C (PLC) (U73122), extracellular-signal regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) (PD089035) and PI-3 kinase (Wortmannin) were not informative. These data suggest that EBV increases migration of AGS cells by a mechanism independent of these autocrine growth factor-induced pathways. Instead, we found that the EBV-infected cells presented increased focal adhesion kinase (FAK) phosphorylation. These findings suggest a role for integrin-mediated signaling in promoting EBV-associated invasiveness.

Acclimation of the European lesser-spotted dogfish Scyliorhinus canicula to reduced environmental salinity [85–70% seawater (SW)] induced a significant diuresis in addition to a significant decrease in plasma osmolality in vivo. The threshold for this diuresis was determined to be 85% SW. Therefore, S. canicula acclimated to 85% SW was selected for further study as a diuretic model in the development of an in situ perfused kidney preparation. The renal role of arginine vasotocin (AVT) in the in situ perfused trunk preparation was investigated. In SW, perfusion of 10−9and 10−10M AVT resulted in a glomerular antidiuresis and decreases in tubular transport maxima for glucose and perfusate flow. In 85% SW, 10−10M AVT had no significant effect on these renal parameters with the exception of transport maxima for glucose and perfusate flow. Tubular parameters remained unchanged by either 10−9or 10−10M AVT. The results demonstrate that the perfused kidney preparation was a viable tool for the investigation of renal parameters in elasmobranch fish and that AVT induced a glomerular antidiuresis.

During wound healing, dermal fibroblasts switch from a migratory, repopulating phenotype to a contractile, matrix-reassembling phenotype. The mechanisms controlling this switch are unknown. A possible explanation is suggested by the finding that chemokines that appear late in wound repair prevent growth factor-induced cell-substratum de-adhesion by blocking calpain activation. In this study, we tested the specific hypothesis that fibroblast contraction of the matrix is promoted by a pro-repair growth factor, epidermal growth factor, and is modulated by calpain-mediated release of adhesions. We employed an isometric force transduction system designed to measure the contraction of a collagen matrix under tension by a population of NR6 fibroblasts transfected with the human epidermal growth factor receptor. By maintaining a fixed level of strain, we could monitor both the initial contraction and subsequent relaxation of the matrix. Epidermal growth factor stimulated a transient, dose-dependent increase in matrix contraction that peaked within 60 minutes and then decayed over the ensuing 3 to 6 hours. Calpain inhibitor I (ALLN) prevented epidermal growth factor-stimulated cell de-adhesion and resulted in a significantly slower decay of matrix contraction, with only a slight decrease of the peak magnitude of contraction. The mitogen-activated protein kinase kinase-1-selective inhibitor PD 98059 that blocks signaling through the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway, required for epidermal growth factor receptor-mediated activation of calpain and de-adhesion, does not significantly affect the magnitude of matrix contraction within minutes of epidermal growth factor addition, but slows the decay similarly to calpain inhibition. Epidermal growth factor receptor signaling thus stimulates the complementary mechanisms of intracellular contractile force generation and calpain-mediated de-adhesion, which are known to coordinately facilitate cell migration. These findings suggest that calpain can act as a functional switch for transmission of intracellular contractile force to the surrounding matrix, with calpain-mediated de-adhesion reducing this transmission and corresponding matrix contraction. Countervailing processes that down-regulate calpain activation can, accordingly, direct the transition of cell function from locomotion to matrix contraction.

During induced cell motility the actin cytoskeleton at the leading edge must undergo constant reorganization. Recently, phosphoinositides have been shown to be central to cytoskeleton-membrane linkages and actin organization and turnover. Epidermal growth factor (EGF) receptor (EGFR)-mediated cell motility requires phospholipase C-gamma (PLCgamma), hydrolysis of phosphoinsotide 4,5-bisphosphate (PIP(2)) and subsequent release of gelsolin. We hypothesized this led to the mobilization of PIP(2)-binding proteins which modify the actin cytoskeleton and thus sought to determine whether the leading edge was a site of active PIP(2) hydrolysis and gelsolin redistribution to cytoskeleton. Herein, we report that during EGF-induced motility, the leading edge's submembranous region constitutes a distinct subcellular locale. The relevant phosphoinositide composition of this space was determined by probing with an antibody to PIP(2) and a green fluorescence protein (GFP)-tagged pleckstrin homology (PH) domain of PLCdelta (GFP-PH) that recognizes both PIP(2) and inositol 1,4,5-trisphosphate (IP(3)). PIP(2) was absent from leading lamellipodia despite an increase in IP(3) generation, suggesting an increase in PIP(2) hydrolysis at the leading edge. Visualized with immunofluorescence, gelsolin preferentially concentrated near the leading edge in a punctate fashion. Examining the Triton X-insoluble actin cytoskeleton fractions, we observe a PLCgamma-dependent increase of gelsolin incorporation upon EGF stimulation. At a molecular level, field emission scanning electron microscopy (FE-SEM) shows that gelsolin incorporates preferentially into the submembranous actin arcs at the leading edge of the lamellipodia. Together these data suggest a model of PIP(2) hydrolysis at the leading edge causing a localized release of PIP(2)-binding proteins-particularly gelsolin-that drives cytoskeletal rearrangement and protrusion.

Induced migration of tumor cells is generally considered to be one critical step in cancer progression to the invasive and metastatic stage. The implicit caveat of studies that show this is that other, unknown, signaling pathways and biophysical events are actually the operative rate-limiting steps, and not motility per se. Thus, to examine the hypothesis that motility is a single, but overall rate-limiting function required for invasion, disparate motility processes need be blocked with concordant effects on tumor invasion. Recently, we and others have described two signaling pathways that are critical to growth factor-induced motility but not mitogenesis. The key molecular switches are phospholipase C-gamma (PLCgamma) and calpain for cytoskeletal reorganization and rear detachment, respectively. We examined this hypothesis in a highly invasive tumor, bladder carcinoma. Three different human tumor cell lines, 253J-B-V, UMUC and T-24, were tested for invasiveness in vitro by transmigration of a Matrigel barrier. Inhibiting PLCgamma with the pharmacologic agent U73122 or the molecular dominant-negative PLCz construct reduced both invasiveness and motility. The same was noted when calpain was blocked using calpain inhibitor I (ALLN). These results demonstrate that one interventional target for limiting invasion is not necessarily an individual motility pathway but rather cell migration per se.

We have shown previously that the ELR-negative CXC chemokines interferon-inducible protein 10, monokine induced by gamma interferon, and platelet factor 4 inhibit epidermal growth factor (EGF)-induced m-calpain activation and thereby EGF-induced fibroblast cell motility (H. Shiraha, A. Glading, K. Gupta, and A. Wells, J. Cell Biol. 146:243-253, 1999). However, how this cross attenuation could be accomplished remained unknown since the molecular basis of physiological m-calpain regulation is unknown. As the initial operative attenuation signal from the CXCR3 receptor was cyclic AMP (cAMP), we verified that this second messenger blocked EGF-induced motility of fibroblasts (55% +/- 4.5% inhibition) by preventing rear release during active locomotion. EGF-induced calpain activation was inhibited by cAMP activation of protein kinase A (PKA), as the PKA inhibitors H-89 and Rp-8Br-cAMPS abrogated cAMP inhibition of both motility and calpain activation. We hypothesized that PKA might negatively modulate m-calpain in an unexpected manner by directly phosphorylating m-calpain. A mutant human large subunit of m-calpain was genetically engineered to negate a putative PKA consensus sequence in the regulatory domain III (ST369/370AA) and was expressed in NR6WT mouse fibroblasts to represent about 30% of total m-calpain in these cells. This construct was not phosphorylated by PKA in vitro while a wild-type construct was, providing proof of the principle that m-calpain can be directly phosphorylated by PKA at this site. cAMP suppressed EGF-induced calpain activity of cells overexpressing a control wild-type human m-calpain (83% +/- 3.7% inhibition) but only marginally suppressed that of cells expressing the PKA-resistant mutant human m-calpain (25% +/- 5.5% inhibition). The EGF-induced motility of the cells expressing the PKA-resistant mutant also was not inhibited by cAMP. Structural modeling revealed that new constraints resulting from phosphorylation at serine 369 would restrict domain movement and help "freeze" m-calpain in an inactive state. These data point to a novel mechanism of negative control of calpain activation, direct phosphorylation by PKA.