Achilles tendon injuries in elite athletes: lessons in pathophysiology from their equine counterparts.

Superficial digital flexor tendon (SDFT) injury in equine athletes is one of the most well-accepted, scientifically supported companion animal models of human disease (i.e., exercise-induced Achilles tendon [AT] injury). The SDFT and AT are functionally and clinically equivalent (and important) energy-storing structures for which no equally appropriate rodent, rabbit, or other analogues exist. Access to equine tissues has facilitated significant advances in knowledge of tendon maturation and aging, determination of specific exercise effects (including early life), and definition of some of the earliest stages of subclinical pathology. Access to human surgical biopsies has provided complementary information on more advanced phases of disease. Importantly, equine SDFT injuries are only a model for acute ruptures in athletes, not the entire spectrum of human tendonopathy (including chronic tendon pain). In both, pathology begins with a potentially prolonged phase of accumulation of (subclinical) microdamage. Recent work has revealed remarkably similar genetic risk factors, including further evidence that tenocyte dysfunction plays an active role. Mice are convenient but not necessarily accurate models for multiple diseases, particularly at the cellular level. Mechanistic studies, including tendon cell responses to combinations of exercise-associated stresses, require a more thorough investigation of cross-species conservation of key stress pathway auditors. Molecular evidence has provided some context for the poor performance of mouse models; equines may provide better systems at this level. The use of horses may be additionally justifiable based on comparable species longevity, lifestyle factors, and selection pressure by similar infectious agents (e.g., herpesviruses) on general cell stress pathway evolution.

Indicators of replicative damage in equine tendon fibroblast monolayers.

BACKGROUND: Superficial digital flexor tendon (SDFT) injuries of horses usually follow cumulative matrix microdamage; it is not known why the reparative abilities of tendon fibroblasts are overwhelmed or subverted. Relevant in vitro studies of this process require fibroblasts not already responding to stresses caused by the cell culture protocols. We investigated indicators of replicative damage in SDFT fibroblast monolayers, effects of this on their reparative ability, and measures that can be taken to reduce it. RESULTS: We found significant evidence of replicative stress, initially observing consistently large numbers of binucleate (BN) cells. A more variable but prominent feature was the presence of numerous gammaH2AX (γH2AX) puncta in nuclei, this being a histone protein that is phosphorylated in response to DNA double-stranded breaks (DSBs). Enrichment for injury detection and cell cycle arrest factors (p53 (ser15) and p21) occurred most frequently in BN cells; however, their numbers did not correlate with DNA damage levels and it is likely that the two processes have different causative mechanisms. Such remarkable levels of injury and binucleation are usually associated with irradiation, or treatment with cytoskeletal-disrupting agents.Both DSBs and BN cells were greatest in subconfluent (replicating) monolayers. The DNA-damaged cells co-expressed the replication markers TPX2/repp86 and centromere protein F. Once damaged in the early stages of culture establishment, fibroblasts continued to express DNA breaks with each replicative cycle. However, significant levels of cell death were not measured, suggesting that DNA repair was occurring. Comet assays showed that DNA repair was delayed in proportion to levels of genotoxic stress. CONCLUSIONS: Researchers using tendon fibroblast monolayers should assess their "health" using γH2AX labelling. Continued use of early passage cultures expressing initially high levels of γH2AX puncta should be avoided for mechanistic studies and ex-vivo therapeutic applications, as this will not be resolved with further replicative cycling. Low density cell culture should be avoided as it enriches for both DNA damage and mitotic defects (polyploidy). As monolayers differing only slightly in baseline DNA damage levels showed markedly variable responses to a further injury, studies of effects of various stressors on tendon cells must be very carefully controlled.

PML: a tumor suppressor that regulates cell fate in mammary gland.

The tumor suppressor promyelocytic leukemia protein (PML) is possibly unique in that its tumor suppressive functions may be attributed to both the protein and the conspicuous nuclear bodies (PML-NBs) that PML builds. Untangling the role of either the protein or its domain in cell fate has been a decade long task which has just received new impetus from developmental biologists. PML appears to play a central role in regulating stem and progenitor cell fate in tissues as diverse as blood, brain and breast. Our studies have uncovered an inverse relationship between the activity of certain Stat transcription factors and PML in controlling normal mammary gland development and the regulation of lineage commitment. Genetic loss of Pml delays differentiation of the milk-producing alveolar cells and disrupts ductal development, defects which may result from a skewing of the progenitor population to favor estrogen receptor positive cells (ERalpha). This is of considerable interest as ERalpha cells are non-cycling in normal breast while promiscuous cell cycle entry is a feature of these cells in breast cancer. These data begin to show the cell types and tissues that are most sensitive to PML dose and provide new perspectives for the regulation of mammary gland development and tumorigenesis.

PML depletion disrupts normal mammary gland development and skews the composition of the mammary luminal cell progenitor pool.

Nuclear domains of promyelocytic leukemia protein (PML) are known to act as signaling nodes in many cellular processes. Although the impact of PML expression in driving cell fate decisions for injured cells is well established, the function of PML in the context of tissue development is less well understood. Here, the in vivo role of PML in developmental processes in the murine mammary gland has been investigated. Data are presented showing that expression of PML is tightly regulated by three members of the Stat family of transcription factors that orchestrate the functional development of the mammary secretory epithelium during pregnancy. Developmental phenotypes were also discovered in the virgin and pregnant Pml null mouse, typified by aberrant differentiation of mammary epithelia with reduced ductal and alveolar development. PML depletion was also found to disturb the balance of two distinct luminal progenitor populations. Overall, it is shown that PML is required for cell lineage determination in bi-potent luminal progenitor cells and that the precise regulation of PML expression is required for functional differentiation of alveolar cells.

Adenovirus type 5 E4 Orf3 protein targets promyelocytic leukaemia (PML) protein nuclear domains for disruption via a sequence in PML isoform II that is predicted as a protein interaction site by bioinformatic analysis.

Human adenovirus type 5 infection causes the disruption of structures in the cell nucleus termed promyelocytic leukaemia (PML) protein nuclear domains or ND10, which contain the PML protein as a critical component. This disruption is achieved through the action of the viral E4 Orf3 protein, which forms track-like nuclear structures that associate with the PML protein. This association is mediated by a direct interaction of Orf3 with a specific PML isoform, PMLII. We show here that the Orf3 interaction properties of PMLII are conferred by a 40 aa residue segment of the unique C-terminal domain of the protein. This segment was sufficient to confer interaction on a heterologous protein. The analysis was informed by prior application of a bioinformatic tool for the prediction of potential protein interaction sites within unstructured protein sequences (predictors of naturally disordered region analysis; PONDR). This tool predicted three potential molecular recognition elements (MoRE) within the C-terminal domain of PMLII, one of which was found to form the core of the Orf3 interaction site, thus demonstrating the utility of this approach. The sequence of the mapped Orf3-binding site on PML protein was found to be relatively poorly conserved across other species; however, the overall organization of MoREs within unstructured sequence was retained, suggesting the potential for conservation of functional interactions.

Death in the AIRE.

When thymic epithelia begin to synthesize peripheral tissue antigens such as insulin, we are seeing the result of autoimmune regulator (AIRE) activity and the workings of central tolerance. AIRE is an extraordinary protein that repatterns the transcriptome of medullary thymic epithelia (mTECs) to produce a stroma decorated with peripheral self-peptides. These peptidic arrays are used to purge self-reactive T cells, thereby averting autoimmunity. We now propose that an inherently cytotoxic event such as global chromatin modification paves the way for AIRE action. This injury stimulus might impose temporal restrictions for the T-cell education process and is endured, at least transiently, by the unique cellular environment provided by the medullary thymic epithelia.

AIRE's CARD revealed, a new structure for central tolerance provokes transcriptional plasticity.

Developing T cells encounter peripheral self-antigens in the thymus in order to delete autoreactive clones. It is now known that the autoimmune regulator protein (AIRE), which is expressed in thymic medullary epithelial cells, plays a key role in regulating the thymic transcription of these peripheral tissue-specific antigens. Mutations in the AIRE gene are associated with a severe multiorgan autoimmune syndrome (APECED), and autoimmune reactivities are manifest in AIRE-deficient mice. Functional AIRE protein is expressed as distinct nuclear puncta, although no structural basis existed to explain their relevance to disease. In addressing the cell biologic basis for APECED, we made the unexpected discovery that an AIRE mutation hot spot lies in a caspase recruitment domain. Combined homology modeling and in vitro data now show how APECED mutations influence the activity of this transcriptional regulator. We also provide novel in vivo evidence for AIRE's association with a global transcription cofactor, which may underlie AIRE's focal, genome-wide, alteration of the transcriptome.

Ataxin-1 fusion partners alter polyQ lethality and aggregation.

Intranuclear inclusion bodies (IBs) are the histopathologic markers of multiple protein folding diseases. IB formation has been extensively studied using fluorescent fusion products of pathogenic polyglutamine (polyQ) expressing proteins. These studies have been informative in determining the cellular targets of expanded polyQ protein as well as the methods by which cells rid themselves of IBs. The experimental thrust has been to intervene in the process of polyQ aggregation in an attempt to alleviate cytotoxicity. However new data argues against the notion that polyQ aggregation and cytotoxicity are inextricably linked processes. We reasoned that changing the protein context of a disease causing polyQ protein could accelerate its precipitation as an IB, potentially reducing its cytotoxicity. Our experimental strategy simply exploited the fact that conjoined proteins influence each others folding and aggregation properties. We fused a full-length pathogenic ataxin-1 construct to fluorescent tags (GFP and DsRed1-E5) that exist at different oligomeric states. The spectral properties of the DsRed1-E5-ataxin-1 transfectants had the additional advantage of allowing us to correlate fluorochrome maturation with cytotoxicity. Each fusion protein expressed a distinct cytotoxicity and IB morphology. Flow cytometric analyses of transfectants expressing the greatest fluorescent signals revealed that the DsRed1-E5-ataxin-1 fusion was more toxic than GFP fused ataxin-1 (31.8+/-4.5% cell death versus 12.85+/-3%), although co-transfection with the GFP fusion inhibited maturation of the DsRed1-E5 fluorochrome and diminished the toxicity of the DsRed1-E5-ataxin-1 fusion. These data show that polyQ driven aggregation can be influenced by fusion partners to generate species with different toxic properties and provide new opportunities to study IB aggregation, maturation and lethality.

Nuclear phospholipase C gamma: punctate distribution and association with the promyelocytic leukemia protein.

The marriage between transducers of cell stress stimuli and their nuclear targets is likely to be achieved in part by some spatial-temporal compartmentalization of the relevant effectors. A candidate compartment for these events is the promyelocytic leukemia nuclear domain (PML-ND), within which are found numerous effectors of damage recognition, repair, and cell death. We predicted that the identification of PML-ND cargo proteins would clarify those biochemical pathways that straddle the recognition of cellular damage and cell fate. We now use mass spectrometry of peptides eluted from PML coprecipitates to demonstrate that the gamma 1 (gamma1) isoform of PLC associates with nuclear PML. Though thought to act primarily in the cytoplasm, we use biochemical fractionation combined with immunocytochemistry to verify the nuclear expression of PLC-gamma1 and its interaction with PML. These are the first data to show an interaction between endogenous levels of a phosphoinositide metabolizing protein and the biophysically labile PML-ND by mass spectrometry and add weight to the view that PML-NDs may act as tumor suppressors by sequestering mitogenic effectors.

Transcriptional repression induces a slowly progressive atypical neuronal death associated with changes of YAP isoforms and p73.

Transcriptional disturbance is implicated in the pathology of polyglutamine diseases, including Huntington's disease (HD). However, it is unknown whether transcriptional repression leads to neuronal death or what forms that death might take. We found transcriptional repression-induced atypical death (TRIAD) of neurons to be distinct from apoptosis, necrosis, or autophagy. The progression of TRIAD was extremely slow in comparison with other types of cell death. Gene expression profiling revealed the reduction of full-length yes-associated protein (YAP), a p73 cofactor to promote apoptosis, as specific to TRIAD. Furthermore, novel neuron-specific YAP isoforms (YAPDeltaCs) were sustained during TRIAD to suppress neuronal death in a dominant-negative fashion. YAPDeltaCs and activated p73 were colocalized in the striatal neurons of HD patients and mutant huntingtin (htt) transgenic mice. YAPDeltaCs also markedly attenuated Htt-induced neuronal death in primary neuron and Drosophila melanogaster models. Collectively, transcriptional repression induces a novel prototype of neuronal death associated with the changes of YAP isoforms and p73, which might be relevant to the HD pathology.

Stress responses of PML nuclear domains are ablated by ataxin-1 and other nucleoprotein inclusions.

The polyglutamine diseases are characterized by expansion of triplet CAG repeats that encode polyglutamine tracts in otherwise unrelated proteins. One plausible explanation for the neurodegeneration of these disorders proposes that inclusions of such proteins sequester other significant nuclear proteins in inactive form. The present study shows that PML protein is sequestered by inclusions of the pathogenic mutant form of the polyglutamine protein ataxin-1 and that this sequestration removes from the nucleus the free 0.2-1 microm diameter PML nuclear domains (PML-NDs), together with at least one of their many cargo proteins (Sp100). The present study demonstrates that this sequestration can be effected equally by another nuclear protein, RED, which lacks a polyglutamine tract, but expresses a polar zipper repeat. The sequestered PML-NDs no longer respond to stress signals (heat shock or ionizing radiation) to which they are normally sensitive. In both cases, there is independent evidence that the cells initiate other responses to their injury (nuclear translocation of heat shock protein or generation of gamma-H2AX-rich nuclear foci, respectively). The data thus provide strong evidence that multiple species of nuclear inclusion functionally sequester PML-NDs. This mechanism is likely to distort cellular responses to injury of many different types.

Interaction between mutant ataxin-1 and PQBP-1 affects transcription and cell death.

PQBP-1 was isolated on the basis of its interaction with polyglutamine tracts. In this study, using in vitro and in vivo assays, we show that the association between ataxin-1 and PQBP-1 is positively influenced by expanded polyglutamine sequences. In cell lines, interaction between the two molecules induces apoptotic cell death. As a possible mechanism underlying this phenomenon, we found that mutant ataxin-1 enhances binding of PQBP-1 to the C-terminal domain of RNA polymerase II large subunit (Pol II). This reduces the level of phosphorylated Pol II and transcription. Our results suggest the involvement of PQBP-1 in the pathology of spinocerebellar ataxia type 1 (SCA1) and support the idea that modified transcription underlies polyglutamine-mediated pathology.