Hans Georg Simon

Interaction of the Extracellular Domain of the Epidermal Growth Factor Receptor with Gangliosides

Ganglioside GM3 inhibits epidermal growth factor (EGF)-dependent cell proliferation in a variety of cell lines. Both in vitro and in vivo, this glycosphingolipid inhibits the kinase activity of the EGF receptor (EGFR). Furthermore, membrane preparations containing EGFR can bind to GM3-coated surfaces. These data suggest that GM3 may interact directly with the EGFR. In this study, the interaction of gangliosides with the extracellular domain (ECD) of the EGFR was investigated. The purified human recombinant ECD from insect cells bound directly to ganglioside GM3. The ganglioside interaction site appears to be distinct from the EGF-binding site. In agreement with previous reports on the effects of specific gangliosides on EGFR kinase activity, the ECD preferentially interacted with GM3. The order of relative binding of other gangliosides investigated was as follows: GM3 ≫ GM2, GD3, GM4 > GM1, GD1a, GD1b, GT1b, GD2, GQ1b > lactosylceramide. These data suggest that NeuAc-lactose is essential for binding and that any sugar substitution reduces binding. In agreement with the specificity of soluble ECD binding to gangliosides, GM3 specifically inhibited EGFR autophosphorylation. Identification of a ganglioside interaction site on the ECD of the EGFR is consistent with the hypothesis that endogenous GM3 may function as a direct modulator of EGFR activity.

A Transitional Extracellular Matrix Instructs Cell Behavior During Muscle Regeneration

Urodele amphibians regenerate appendages through the recruitment of progenitor cells into a blastema that rebuilds the lost tissue. Blastemal formation is accompanied by extensive remodeling of the extracellular matrix. Although this remodeling process is important for appendage regeneration, it is not known whether the remodeled matrix directly influences the generation and behavior of blastemal progenitor cells. By integrating in vivo 3-dimensional spatiotemporal matrix maps with in vitro functional time-lapse imaging, we show that key components of this dynamic matrix, hyaluronic acid, tenascin-C and fibronectin, differentially direct cellular behaviors including DNA synthesis, migration, myotube fragmentation and myoblast fusion. These data indicate that both satellite cells and fragmenting myofibers contribute to the regeneration blastema and that the local extracellular environment provides instructive cues for the regenerative process. The fact that amphibian and mammalian myoblasts exhibit similar responses to various matrices suggests that the ability to sense and respond to regenerative signals is evolutionarily conserved.

A Dynamic Spatiotemporal Extracellular Matrix Facilitates Epicardial-Mediated Vertebrate Heart Regeneration

Unlike humans, certain adult vertebrates such as newts and zebrafish possess extraordinary abilities to functionally regenerate lost appendages and injured organs, including cardiac muscle. Here, we present new evidence that a remodeled extracellular matrix (ECM) directs cell activities essential for cardiac muscle regeneration. Comprehensive mining of DNA microarrays and Gene Ontology term enrichment analyses for regenerating newt and zebrafish hearts revealed that distinct ECM components and ECM-modifying proteases are among the most significantly enriched genes in response to local injury. In contrast, data analyses for mammalian cardiac injury models indicated that inflammation and metabolic processes are the most significantly activated gene groups. In the regenerating newt heart, we show dynamic spatial and temporal changes in tenascin-C, hyaluronic acid, and fibronectin ECM distribution as early as 3 days postamputation. Linked to distinct matrix remodeling, we demonstrate a myocardium-wide proliferative response and radial migration of progenitor cells. In particular, we report dramatic upregulation of a regeneration-specific matrix in the epicardium that precedes the accumulation and migration of progenitor cells. For the first time, we show that the regenerative ECM component tenascin-C significantly increases newt cardiomyocyte cell cycle reentry in vitro. Thus, the engineering of nature-tested extracellular matrices may provide new strategic opportunities for the enhancement of regenerative responses in mammals.

Nucleocytoplasmic functions of the PDZ‐LIM protein family: new insights into organ development

Recent work on the PDZ‐LIM protein family has revealed that it has important activities at the cellular level, mediating signals between the nucleus and the cytoskeleton, with significant impact on organ development. We review and integrate current knowledge about the PDZ‐LIM protein family and propose a new functional role, sequestering nuclear factors in the cytoplasm. Characterized by their PDZ and LIM domains, the PDZ‐LIM family is comprised of evolutionarily conserved proteins found throughout the animal kingdom, from worms to humans. Combining two functional domains in one protein, PDZ‐LIM proteins have wide‐ranging and multi‐compartmental cell functions during development and homeostasis. In contrast, misregulation can lead to cancer formation and progression. New emerging roles include interactions with integrins, T‐box transcription factors, and receptor tyrosine kinases. Facilitating the assembly of protein complexes, PDZ‐LIM proteins can act as signal modulators, influence actin dynamics, regulate cell architecture, and control gene transcription.

Biochemical and mechanical environment cooperatively regulate skeletal muscle regeneration

During forelimb regeneration in the newt Notophthalmus viridescens, the dynamic expression of a transitional matrix rich in hyaluronic acid, tenascin‐C, and fibronectin controls muscle cell behavior in vivo and in vitro. However, the influence of extracellular matrix (ECM) remodeling on tissue stiffness and the cellular response to mechanical variations during regeneration was unknown. By measuring the transverse stiffness of tissues in situ, we found undifferentiated regenerative blastemas were less stiff than differentiated stump muscle (13.3±1.6 vs. 16.6±1.2 kPa). To directly determine how ECM and stiffness combine to affect skeletal muscle fragmentation, migration, and fusion, we coated silicone‐based substrates ranging from 2 to 100 kPa with matrices representative of transitional (tenascin‐C and fibronectin) and differentiated environments (laminin and Matrigel). Using live‐cell imaging, we found softer tenascin‐C‐coated substrates significantly enhanced migration and fragmentation of primary newt muscle cells. In contrast, stiffer substrates coated with laminin, Matrigel, or fibronectin increased differentiation while suppressing migration and fragmentation. These data support our in vivo observations that a transitional matrix of reduced stiffness regulates muscle plasticity and progenitor cell recruitment into the regenerative blastema. These new findings will enable the determination of how biochemical and mechanical cues from the ECM control genetic pathways that drive regeneration.—Calve, S., Simon, H.‐G. Biochemical and mechanical environment cooperatively regulate skeletal muscle regeneration. FASEB J. 26, 2538‐2545 (2012). www.fasebj.org

Pdlim7 (LMP4) regulation of Tbx5 specifies zebrafish heart atrio-ventricular boundary and valve formation.

Tbx5 is involved in congenital heart disease, however, the mechanisms leading to organ malformation are greatly unknown. We hypothesized a model by which the Tbx5 binding protein Pdlim7 controls nuclear/cytoplasmic shuttling and function of the transcription factor. Using the zebrafish, we present in vivo significance for an essential role of Tbx5/Pdlim7 protein interaction in the regulation of cardiac formation. Knock-down of Pdlim7 results in a non-looped heart, strikingly reminiscent of the tbx5 heartstrings mutant phenotype. However, while misregulation of Pdlim7 and Tbx5 produce similar aberrant cardiac morphology, molecular and histological analysis uncovered that the Pdlim7 and Tbx5 cardiac phenotypes are due to opposite effects on valve development. Loss of Pdlim7 function causes no valve tissue to develop while lack of Tbx5 results in increased valve tissue. These opposing defects are evident before valve formation and are the result of distinct gene misregulation during specification of the atrio-ventricular (AV) boundary. We show that Pdlim7/Tbx5 interactions affect the expression of Tbx5 target genes nppa and tbx2b at the AV boundary, and their domains of misexpression directly correlate with the identified valve defects. These studies demonstrate that controlling the correct balance of Tbx5 activity is crucial for the specification of the AV boundary and valve formation.

T-box genes and the formation of vertebrate forelimb- and hindlimb specific pattern.

Limb patterning is thought to be a multistep process involving specification of the limb fields, establishment of defined signaling centers that globally inform cells of their position, interpretation of positional signals, and regulated growth and differentiation of the limb structures. Great progress has been made over the past few years in identifying the molecular players that control limb outgrowth and patterning, in particular, how the limb axes are specified. However, the molecular mechanism for determination of the morphological and functional differences between forelimbs and hindlimbs has remained elusive. The recent identification of a series of limb-specific transcription factors has now provided excellent candidates for such upstream regulators of limb identity, and has allowed new insights into the regulatory network of making a hand or a foot.

An Evolutionarily Conserved Nuclear Export Signal Facilitates Cytoplasmic Localization of the Tbx5 Transcription Factor

ABSTRACT During cardiac development, the T-box transcription factor Tbx5 displays dynamic changes in localization from strictly nuclear to both nuclear and cytoplasmic to exclusively cytoplasmic along the actin cytoskeleton in cells coexpressing its binding protein LMP4. Although nuclear localization signals (NLSs) have been described, the mechanism by which Tbx5 exits the nucleus remained elusive. Here, we describe for Tbx5 a nuclear export signal (NES) that is recognized by the CRM1 export protein. Site-directed mutagenesis of a critical amino acid(s) within this sequence determined the functionality of this NES. Confocal localization studies and luciferase transcriptional reporter assays with NES mutant Tbx5 forms demonstrated retention in the nucleus, regardless of the presence of LMP4. Coimmunoprecipitation and pharmacological interference studies demonstrated a direct interaction between Tbx5 and CRM1, revealing that Tbx5 is using the CRM1 pathway for nuclear export. In addition to Tbx5, we identified NESs in all T-box proteins and demonstrated interaction of the family members Tbx3 and Brachyury with the CRM1 exporter, suggesting general significance. This first demonstration of evolutionarily conserved NESs in all T-box proteins in conjunction with NLSs indicates a primordial function of T-box proteins to dynamically shuttle between nuclear and cytoplasmic compartments of the cell.

Bone Formation During Forelimb Regeneration: A Microtomography (MicroCT) Analysis

In our study of bone regeneration in the forelimbs of mature newts (Notophthalmus viridescens), we used noninvasive X‐ray microtomography (microCT) to image regenerating limbs from 37 to 85 days and matching (contralateral) controls. We compared the patterns of regenerated and existing (nonregenerated) bone, investigating in particular the onset of mineralization of specific bones, the level of mineral present, and the lengths of the different bones. Overall, we find that the missing limb skeletal elements are restored in a proximal‐to‐distal direction, which reiterates the developmental patterning program. However, in contrast to this proximal–distal sequence, the portion of the humerus distal to the amputation site fails to ossify in synchrony with the regenerating radius and ulna. This finding suggests that the replacement of cartilage with mineralized bone close to the amputation site is delayed with respect to other regenerating skeletal elements. Developmental Dynamics 226:410–417, 2003.

Multi-tissue microarray analysis identifies a molecular signature of regeneration.

The inability to functionally repair tissues that are lost as a consequence of disease or injury remains a significant challenge for regenerative medicine. The molecular and cellular processes involved in complete restoration of tissue architecture and function are expected to be complex and remain largely unknown. Unlike humans, certain salamanders can completely regenerate injured tissues and lost appendages without scar formation. A parsimonious hypothesis would predict that all of these regenerative activities are regulated, at least in part, by a common set of genes. To test this hypothesis and identify genes that might control conserved regenerative processes, we performed a comprehensive microarray analysis of the early regenerative response in five regeneration-competent tissues from the newt Notophthalmus viridescens. Consistent with this hypothesis, we established a molecular signature for regeneration that consists of common genes or gene family members that exhibit dynamic differential regulation during regeneration in multiple tissue types. These genes include members of the matrix metalloproteinase family and its regulators, extracellular matrix components, genes involved in controlling cytoskeleton dynamics, and a variety of immune response factors. Gene Ontology term enrichment analysis validated and supported their functional activities in conserved regenerative processes. Surprisingly, dendrogram clustering and RadViz classification also revealed that each regenerative tissue had its own unique temporal expression profile, pointing to an inherent tissue-specific regenerative gene program. These new findings demand a reconsideration of how we conceptualize regenerative processes and how we devise new strategies for regenerative medicine.