Faculty Bibliography

The COP9 signalosome inhibits the activity of Cullin-RING E3 ubiquitin ligases by removing Nedd8 modifications from their Cullin subunits. Neddylation renders these complexes catalytically active, but deneddylation is also necessary for them to exchange adaptor subunits and avoid auto-ubiquitination. Although deneddylation is thought to be the primary function of the COP9 signalosome, additional activities have been ascribed to some of its subunits. We recently showed that COP9 subunits protect the transcriptional repressor and tumor suppressor Capicua from two distinct modes of degradation. Deneddylation by the COP9 signalosome inactivates a Cullin 1 complex that ubiquitinates Capicua following its phosphorylation by MAP kinase in response to Epidermal Growth Factor Receptor signaling. The CSN1b subunit also stabilizes unphosphorylated Capicua to control its basal level, independently of the deneddylase function of the complex. Here we further examine the importance of deneddylation for COP9 functions in vivo. We use an uncleavable form of Nedd8 to show that preventing deneddylation does not reproduce the effects of loss of COP9. In contrast, in the presence of COP9, conjugation to uncleavable Nedd8 renders Cullins unable to promote the degradation of their substrates. Our results suggest that irreversible neddylation prolongs COP9 binding to and inhibition of Cullin-based ubiquitin ligases.

Cells release extracellular vesicles (EVs) that mediate intercellular communication and repair damaged membranes. Despite the pleiotropic functions of EVs in vitro, their in vivo function is debated, largely because it is unclear how to induce or inhibit their formation. In particular, the mechanisms of EV release by plasma membrane budding or ectocytosis are poorly understood. We previously showed that TAT-5 phospholipid flippase activity maintains the asymmetric localization of the lipid phosphatidylethanolamine (PE) in the plasma membrane and inhibits EV budding by ectocytosis in Caenorhabditis elegans However, no proteins that inhibit ectocytosis upstream of TAT-5 were known. Here, we identify TAT-5 regulators associated with retrograde endosomal recycling: PI3Kinase VPS-34, Beclin1 homolog BEC-1, DnaJ protein RME-8, and the uncharacterized Dopey homolog PAD-1. PI3Kinase, RME-8, and semiredundant sorting nexins are required for the plasma membrane localization of TAT-5, which is important to maintain PE asymmetry and inhibit EV release. PAD-1 does not directly regulate TAT-5 localization, but is required for the lipid flipping activity of TAT-5. PAD-1 also has roles in endosomal trafficking with the GEF-like protein MON-2, which regulates PE asymmetry and EV release redundantly with sorting nexins independent of the core retromer. Thus, in addition to uncovering redundant intracellular trafficking pathways, our study identifies additional proteins that regulate EV release. This work pinpoints TAT-5 and PE as key regulators of plasma membrane budding, further supporting the model that PE externalization drives ectocytosis.

The assembly of functional neuronal circuits requires growth cones to extend in defined directions and recognize the correct synaptic partners. Homophilic adhesion between vertebrate Sidekick proteins promotes synapse formation between retinal neurons involved in visual motion detection. We show here that that Drosophila Sidekick accumulates in specific synaptic layers of the developing motion detection circuit and is necessary for normal optomotor behavior. Sidekick is required in photoreceptors, but not their target lamina neurons, to promote the alignment of lamina neurons into columns and subsequent sorting of photoreceptor axons into synaptic modules based on their precise spatial orientation. Sidekick is also localized to the dendrites of the direction-selective T4 and T5 cells, and is expressed in some of their presynaptic partners. In contrast to its vertebrate homologues, Sidekick is not essential for T4 and T5 to direct their dendrites to the appropriate layers or to receive synaptic contacts. These results illustrate a conserved requirement for Sidekick proteins to establish visual motion detection circuits that is achieved through distinct cellular mechanisms in Drosophila and vertebrates.

PURPOSE/OBJECTIVE:The purpose of this study was to quantify the regional histology of the long head of the biceps tendon (LHBT) and compare the histopathology present to clinical findings in patients with rotator cuff tears and SLAP lesions. METHODS:Prospectively enrolled patients undergoing an open subpectoral LHBT tenodesis in the setting of a rotator cuff (RTC) tear or SLAP lesion. Perioperative data were collected and the excised LHBT was analyzed by a fellowship trained pathologist. Tendons were sectioned into proximal (biceps anchor), middle (bicipital groove), and distal (myotendinous junction) portions. Sections were stained with Movat's pentachrome stain and digitized for analysis. Comparisons were made between the histologic findings present in the setting of a rotator cuff tear with those seen in the setting of a SLAP tear. RESULTS:39 tendons were analyzed: 20 from patients with SLAP lesions (mean age of 44.7 years, range 23-60 years) and 19 from patients with rotator cuff tears (mean age of 58.7 years, range 43-71). Patients with the most pathologic tendons in the bicipital groove were significantly older (59.4 vs. 50.4 years; p < 0.05), reported higher pre-operative VAS scores (6.6 vs. 5.0; p < 0.02), and demonstrated lower pre-operative ASES scores (41.6 vs. 50.7; p < 0.05). The RTC group showed significantly more mucinous degeneration at both the proximal (p < 0.03) and the middle (p < 0.01) tendon portions compared to the SLAP group. In both groups, the portions of proximal tendon showed significantly (p < 0.05) more mucinous degeneration than distal portions. CONCLUSION/CONCLUSIONS:Regional histologic differences exist in the LHBT. Rotator cuff patients showed the most degenerated tendon in the bicipital groove and these patients tended to be older and have higher VAS and lower ASES scores. Surgeons should consider performing a subpectoral biceps tenodesis as the bicipital groove portion of the tendon may be very degenerated, especially in patients with rotator cuff disease. Additional research is warranted to distinguish whether treating the biceps differently in distinct geographic regions affects patient outcomes. LEVEL OF EVIDENCE/METHODS:II.

Mechanical loading is an important aspect of post-surgical fracture care. The timing of load application relative to the injury event may differentially regulate repair depending on the stage of healing. Here, we used a novel mechanobiological model of cortical defect repair that offers several advantages including its technical simplicity and spatially confined repair program, making effects of both physical and biological interventions more easily assessed. Using this model, we showed that daily loading (5N peak load, 2Hz, 60 cycles, 4 consecutive days) during hematoma consolidation and inflammation disrupted the injury site and activated cartilage formation on the periosteal surface adjacent to the defect. We also showed that daily loading during the matrix deposition phase enhanced both bone and cartilage formation at the defect site, while loading during the remodeling phase resulted in an enlarged woven bone regenerate. All loading regimens resulted in abundant cellular proliferation throughout the regenerate and fibrous tissue formation directly above the defect demonstrating that all phases of cortical defect healing are sensitive to physical stimulation. Stress was concentrated at the edges of the defect during exogenous loading, and finite element (FE)-modeled longitudinal strain (εzz) values along the anterior and posterior borders of the defect (~2200με) was an order of magnitude larger than strain values on the proximal and distal borders (~50-100με). It is concluded that loading during the early stages of repair may impede stabilization of the injury site important for early bone matrix deposition, whereas loading while matrix deposition and remodeling are ongoing may enhance stabilization through the formation of additional cartilage and bone.

It has been shown that dominant tolerance, namely in transplantation, requires Foxp3+ regulatory T cells. Although most tolerance-inducing regimens rely on regulatory T cells, we found that induction of tolerance to proteins in aluminum hydroxide can be achieved in Foxp3-deficient mice using nondepleting anti-CD4 Abs. This type of tolerance is Ag specific, and tolerant mice retain immune competence to respond to unrelated Ags. We demonstrated with chicken OVA-specific TCR-transgenic mice that the same tolerizing protocol (CD4 blockade) and the same target Ag (OVA) achieves Foxp3-dependent transplantation tolerance to OVA-expressing skin grafts, but Foxp3-independent tolerance when the Ag is provided as OVA-aluminum hydroxide. In the latter case, we found that tolerance induction triggered recessive mechanisms leading to elimination of effector cells and, simultaneously, a dominant mechanism associated with the emergence of an anergic and regulatory CTLA-4+IL-2lowFoxp3- T cell population, where the tolerance state is IL-10 dependent. Such Foxp3-independent mechanisms can improve the efficacy of tolerance-inducing protocols.

Latent transforming growth factor β (TGFβ)-binding proteins (LTBPs) are important for the secretion, activation, and function of mature TGFβ, especially so in cancer cell physiology. However, specific roles of the LTBPs remain understudied in the context of the primary tumor microenvironment. Herein, we investigated the role of LTBP3 in the distinct processes involved in cancer metastasis. By using three human tumor cell lines of different tissue origin (epidermoid HEp-3 and prostate PC-3 carcinomas and HT-1080 fibrosarcoma) and several metastasis models conducted in both mammalian and avian settings, we show that LTBP3 is involved in the early dissemination of primary cancer cells, namely in the intravasation step of the metastatic cascade. Knockdown of LTBP3 in all tested cell lines led to significant inhibition of tumor cell intravasation, but did not affect primary tumor growth. LTBP3 was dispensable in the late steps of carcinoma cell metastasis that follow tumor cell intravasation, including vascular arrest, extravasation, and tissue colonization. However, LTBP3 depletion diminished the angiogenesis-inducing potential of HEp-3 cells in vivo, which was restorable by exogenous delivery of LTBP3 protein. A similar compensatory approach rescued the dampened intravasation of LTBP3-deficient HEp-3 cells, suggesting that LTBP3 regulates the induction of the intravasation-supporting angiogenic vasculature within developing primary tumors. Using our recently developed microtumor model, we confirmed that LTBP3 loss resulted in the development of intratumoral vessels with an abnormal microarchitecture incompatible with efficient intravasation of HEp-3 carcinoma cells. Collectively, these findings demonstrate that LTBP3 represents a novel oncotarget that has distinctive functions in the regulation of angiogenesis-dependent tumor cell intravasation, a critical process during early cancer dissemination. Our experimental data are also consistent with the survival prognostic value of LTBP3 expression in early-stage head and neck squamous cell carcinomas, further indicating a specific role for LTBP3 in cancer progression toward metastatic disease.

Polycomb group proteins regulate self-renewal and differentiation in many stem cell systems. When assembled into two canonical complexes, PRC1 and PRC2, they sequentially deposit H3K27me3 and H2AK119ub histone marks and establish repressive chromatin, referred to as Polycomb domains. Non-canonical PRC1 complexes retain RING1/RNF2 E3-ubiquitin ligases but have unique sets of accessory subunits. How these non-canonical complexes recognize and regulate their gene targets remains poorly understood. Here, we show that the BCL6 co-repressor (BCOR), a member of the PRC1.1 complex, is critical for maintaining primed pluripotency in human embryonic stem cells (ESCs). BCOR depletion leads to the erosion of Polycomb domains at key developmental loci and the initiation of differentiation along endoderm and mesoderm lineages. The C terminus of BCOR regulates the assembly and targeting of the PRC1.1 complex, while the N terminus contributes to BCOR-PRC1.1 repressor function. Our findings advance understanding of Polycomb targeting and repression in ESCs and could apply broadly across developmental systems.

The latent transforming growth factor (TGF) β binding proteins (LTBP) are crucial mediators of TGFβ function, as they control growth factor secretion, matrix deposition, presentation and activation. Deficiencies in specific LTBP isoforms yield discrete phenotypes representing defects in bone, lung and cardiovascular development mediated by loss of TGFβ signaling. Additional phenotypes represent loss of unique TGFβ-independent features of LTBP effects on elastogenesis and microfibril assembly. Thus, the LTBPs act as sensors for the regulation of both growth factor activity and matrix function.

Large excisional wounds in mice prominently regenerate new hair follicles (HFs) and fat, yet humans are deficient for this regenerative behavior. Currently, wound-induced regeneration remains a clinically desirable, but only partially understood phenomenon. We show that large excisional wounds in rats, across seven strains fail to regenerate new HFs. We compared wound transcriptomes between mice and rats at the time of scab detachment, which coincides with the onset of HF regeneration in mice. In both species, wound dermis and epidermis share core dermal and epidermal transcriptional programs respectively, yet prominent inter-species differences exist. Compared to mice, rat epidermis expresses distinct transcriptional and epigenetic factors, markers of epidermal repair, hyperplasia, and inflammation, and lower levels of WNT signaling effectors and regulators. When recombined on the surface of excisional wounds with vibrissa dermal papillae, partial-thickness skin grafts containing distal pelage HF segments, but not interfollicular epidermis, readily regenerated new vibrissa-like HFs. Together, our findings establish rats as a non-regenerating rodent model for excisional wound healing and suggest that low epidermal competence and associated transcriptional profile may contribute to its regenerative deficiency. Future comparison between rat and mouse may lend further insight into the mechanism of wounding-induced regeneration and causes for its deficit.