Justine C. Lee

NF-κB protects from the lysosomal pathway of cell death

The programme of gene expression induced by RelA/NF‐κB transcription factors is critical to the control of cell survival. Ligation of ‘death receptors’ such as tumor necrosis factor receptor 1 (TNF‐R1) triggers apoptosis, as well as NF‐κB, which counteracts this process by activating the transcription of anti‐apoptotic genes. In addition to activating caspases, TNF‐R1 stimulation causes the release of cathepsins, most notably cathepsin B, from the lysosome into the cytoplasm where they induce apoptosis. Here we report a mechanism by which NF‐κB protects cells against TNF‐α‐induced apoptosis: inhibition of the lysosomal pathway of apoptosis. NF‐κB can protect cells from death after TNF‐R1 stimulation, by extinguishing cathepsin B activity in the cytosol. This activity of NF‐κB is mediated, at least in part, by the upregulation of Serine protease inhibitor 2A (Spi2A), a potent inhibitor of cathepsin B. Indeed, Spi2A can substitute for NF‐κB in suppressing the induction of cathepsin B activity in the cytosol. Thus, inhibition of cathepsin B by Spi2A is a mechanism by which NF‐κB protects cells from lysosome‐mediated apoptosis.

The death effector domain protein family

Apoptosis signaling is regulated and executed by specialized proteins that often carry protein/protein interaction domains. One of these domains is the death effector domain (DED) that is predominantly found in components of the death-inducing signaling complex, which forms at the members of the death receptor family following their ligation. Both proapoptotic- and antiapoptotic-DED-containing proteins have been identified, which makes these proteins exquisitely suited to the regulation of apoptosis. Aside from their pivotal role in the control of the apoptotic program, DED-containing proteins have recently been demonstrated to exert their influence on other cellular processes as well, including cell proliferation. These data highlight the multiple roles for the members of this family, suggesting that they are suited to control both life and death decisions of cells. Additionally, because they can act proapoptotically, antiapoptotically, or in the regulation of the cell cycle, this family of proteins may be excellent candidates for cancer therapy targets.

DEDD regulates degradation of intermediate filaments during apoptosis.

Apoptosis depends critically on regulated cytoskeletal reorganization events in a cell. We demonstrate that death effector domain containing DNA binding protein (DEDD), a highly conserved and ubiquitous death effector domain containing protein, exists predominantly as mono- or diubiquitinated, and that diubiquitinated DEDD interacts with both the K8/18 intermediate filament network and pro–caspase-3. Early in apoptosis, both cytosolic DEDD and its close homologue DEDD2 formed filaments that colocalized with and depended on K8/18 and active caspase-3. Subsequently, these filamentous structures collapsed into intracellular inclusions that migrated into cytoplasmic blebs and contained DEDD, DEDD2, active caspase-3, and caspase-3–cleaved K18 late in apoptosis. Biochemical studies further confirmed that DEDD coimmunoprecipitated with both K18 and pro–caspase-3, and kinetic analyses placed apoptotic DEDD staining prior to caspase-3 activation and K18 cleavage. In addition, both caspase-3 activation and K18 cleavage was inhibited by expression of DEDDΔNLS1-3, a cytosolic form of DEDD that cannot be ubiquitinated. Finally, siRNA mediated DEDD knockdown cells exhibited inhibition of staurosporine-induced DNA degradation. Our data suggest that DEDD represents a novel scaffold protein that directs the effector caspase-3 to certain substrates facilitating their ordered degradation during apoptosis.

Regulation of apoptosis by ubiquitination

Summary:  Cell elimination through apoptosis, or programmed cell death, is an evolutionarily conserved central tenet of biology from embryological development to immune homeostasis. While many of the apoptotic signaling pathways have been elucidated, the relationship between ubiquitin and apoptosis is only beginning to be defined. In the past decade, many reports of polyubiquitin conjugation of key pro‐ and anti‐apoptotic molecules have characterized ubiquitin as an essential regulatory modification targeting proteins for proteasomal degradation. However, recent work relating monoubiquitination and nonclassical polyubiquitin conjugation to apoptotic molecules has added an additional level of diversity to the role of ubiquitin in apoptotic regulation beyond degradation. This review focuses on the direct effects of ubiquitination on apoptosis‐signaling molecules.

Intermediate filaments control the intracellular distribution of caspases during apoptosis.

Caspases are responsible for a cascade of events controlling the disassembly of apoptotic cells. We now demonstrate that caspase-9 is activated at an early stage of apoptosis in epithelial cells and all its detectable, catalytically active large subunits (both the p35 and p37) are concentrated on cytokeratin fibrils. Immunolabeling of distinctive neoepitopes, exposed by cleavage of procaspase-9 at either Asp315 or Asp330, was co-localized on these fibrils with active caspase-3, caspase-cleaved cytokeratin-18, death-effector-domain containing DNA-binding protein and ubiquitin. Cytokeratin filaments may thus provide a scaffold whereby active subunits of caspase-9 can activate caspase-3 which, in turn, can activate more caspase-9 so forming an amplification loop to facilitate cleavage of cytokeratin-18, disruption of the cytoskeleton and the ensuing formation of cytoplasmic inclusions. These inclusions, formed from the collapse of fibrils, together with their associated components, also contain ubiquitinated proteins, vimentin, heat-shock protein 72, and tumor necrosis factor receptor type-1-associated death domain protein. Many of their constituents, including active caspases, remain sequestered within these inclusions, even after detergent treatment and isolation. Thus, such inclusions do not merely accumulate disrupted cytokeratins but also sequestrate potentially noxious proteins that could injure healthy neighboring cells.

Osteogenesis on nanoparticulate mineralized collagen scaffolds via autogenous activation of the canonical BMP receptor signaling pathway

Skeletal regenerative medicine frequently incorporates deliverable growth factors to stimulate osteogenesis. However, the cost and side effects secondary to supraphysiologic dosages of growth factors warrant investigation of alternative methods of stimulating osteogenesis for clinical utilization. In this work, we describe growth factor independent osteogenic induction of human mesenchymal stem cells (hMSCs) on a novel nanoparticulate mineralized collagen glycosaminoglycan scaffold (MC-GAG). hMSCs demonstrated elevated osteogenic gene expression and mineralization on MC-GAG with minimal to no effect upon addition of BMP-2 when compared to non-mineralized scaffolds (Col-GAG). To investigate the intracellular pathways responsible for the increase in osteogenesis, we examined the canonical and non-canonical pathways downstream from BMP receptor activation. Constitutive Smad1/5 phosphorylation with nuclear translocation occurred on MC-GAG independent of BMP-2, whereas Smad1/5 phosphorylation depended on BMP-2 stimulation on Col-GAG. When non-canonical BMPR signaling molecules were examined, ERK1/2 phosphorylation was found to be decreased in MC-GAG but elevated in Col-GAG. No differences in Smad2/3 or p38 activation were detected. Collectively, these results demonstrated that MC-GAG scaffolds induce osteogenesis without exogenous BMP-2 addition via endogenous activation of the canonical BMP receptor signaling pathway.

Autologous immediate cranioplasty with vascularized bone in high-risk composite cranial defects.

Background: Composite cranial defects in the setting of infection, irradiation, or cerebrospinal fluid leak present a significant risk for devastating neurologic sequelae. Such defects require soft-tissue coverage and skeletal reconstruction that can withstand the hostile environment of a precarious wound. Methods: Patients with high-risk composite cranial defects treated with free flap reconstruction containing a vascularized osseous component from 2003 to 2012 were reviewed retrospectively. Results: Fourteen patients received autologous vascularized cranioplasties between 2003 and 2012 with a mean age of 55.7 years and a mean follow-up of 14.1 months. Preoperatively, all patients had infection, irradiation, cerebrospinal fluid leak, or a combination thereof. Thirteen patients (92.9 percent) were reoperative cases for recurrent tumor, infection, or both. Six patients (42.9 percent) failed previous reconstructive procedures. Tissue biopsy–proven infection was present in 10 patients (71.4 percent) with calvarial osteomyelitis, both osteomyelitis and meningitis, or scalp soft-tissue infection only. Nine patients (64.3 percent) suffered from malignancy and six of these patients were irradiated preoperatively. Cranioplasty was achieved as part of a chimeric free flap using rib, scapula, both rib and scapula, or ilium. Vascularized duraplasty using serratus anterior fascia as a component of the chimeric flap was performed in three patients. No flap losses occurred and all patients had resolution of infection. Conclusions: Soft-tissue and skeletal restoration are the two critical components of composite cranial reconstruction. The authors report outcomes of the largest series of one-stage immediate cranioplasty consisting of autologous soft tissue and vascularized bone in high-risk composite cranial wounds and suggest its application in defects associated with compromised wound beds. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Primary sternal closure with titanium plate fixation: plastic surgery effecting a paradigm shift.

Background: Postoperative mediastinitis is a serious and potentially lethal complication from cardiac surgery. Although postoperative mediastinitis cannot be reliably predicted, a number of preoperative and intraoperative risk factors have been defined by previous work. The authors now present their cumulative experience with primary sternal fixation of high-risk patients as one preventative measure. Methods: A retrospective review from July of 2000 to October of 2006 was performed on 750 patients who had at least three established risk factors for postoperative mediastinitis and received primary titanium plate sternal fixation. Patients were followed for a minimum of 6 weeks and monitored for pain, instability, wound breakdown, and plate migration. Results: Rigid plate fixation was completed at the end of the primary cardiac surgical procedure in all 750 patients. Sternal dehiscence occurred in 18 patients (2.4 percent), necessitating reexploration. Four of these patients developed postoperative mediastinitis and had other significant comorbidities, such as ongoing inflammatory breast cancer or pneumonia, that were beyond the typical risk factors identified for developing mediastinitis. Successful sternal fixation was therefore accomplished in 732 patients (97.6 percent). Despite changes in instrumentation and technique, this approach was adopted by the cardiac surgical team consistently after an initial mentoring and training period by the plastic surgeons. Conclusions: Primary sternal fixation is a simple and reliable method for prevention of postoperative mediastinitis development in high-risk patients. This technique, conceptualized by plastic surgeons, is now being implemented by cardiac surgeons in increasing numbers. This demonstrates the ability for plastic surgery to initiate a paradigm shift in other fields of medicine and to decrease the complications that primarily affect our practice.

Optimizing Collagen Scaffolds for Bone Engineering: Effects of Cross-linking and Mineral Content on Structural Contraction and Osteogenesis.

Introduction: Osseous defects of the craniofacial skeleton occur frequently in congenital, posttraumatic, and postoncologic deformities. The field of scaffold-based bone engineering emerged to address the limitations of using autologous bone for reconstruction of such circumstances. In this work, the authors evaluate 2 modifications of three-dimensional collagen-glycosaminoglycan scaffolds in an effort to optimize structural integrity and osteogenic induction. Methods: Human mesenchymal stem cells (hMSCs) were cultured in osteogenic media on nonmineralized collagen-glycosaminoglycan (C-GAG) and nanoparticulate mineralized collagen-glycosaminoglycan (MC-GAG) type I scaffolds, in the absence and presence of cross-linking. At 1, 7, and 14 days, mRNA expression was analyzed using quantitative real-time -reverse-transcriptase polymerase chain reaction for osteocalcin (OCN) and bone sialoprotein (BSP). Structural contraction was measured by the ability of the scaffolds to maintain their original dimensions. Mineralization was detected by microcomputed tomographic (micro-CT) imaging at 8 weeks. Statistical analyses were performed with Student t-test. Results: Nanoparticulate mineralization of collagen-glycosaminoglycan scaffolds increased expression of both OCN and BSP. Cross-linking of both C-GAG and MC-GAG resulted in decreased osteogenic gene expression; however, structural contraction was significantly decreased after cross-linking. Human mesenchymal stem cells-directed mineralization, detected by micro-CT, was increased in nanoparticulate mineralized scaffolds, although the density of mineralization was decreased in the presence of cross-linking. Conclusions: Optimization of scaffold material is an essential component of moving toward clinically translatable engineered bone. Our current study demonstrates that the combination of nanoparticulate mineralization and chemical cross-linking of C-GAG scaffolds generates a highly osteogenic and structurally stable scaffold.

Fusing DEDD with ubiquitin changes its intracellular localization and apoptotic potential

DEDD, a highly conserved and ubiquitous death effector domain containing protein, exists in non, mono, and diubiquitinated forms. We previously reported that endogenous unmodified DEDD is only found in nucleoli and that mono- and diubiquitinated DEDD associate with caspase-3 in the cytosol suggesting that ubiquitination may be important to the apoptosis regulating functions of DEDD in the cytosol. We now demonstrate that many of its 16 lysine residues can serve as alternative acceptors for ubiquitination to maintain the monoubiquitination status of DEDD. A central region in DEDD (amino acids 109–305) outside the death effector domain was found to be essential for ubiquitination and/or the docking of the ubiquitination machinery. Fusion of ubiquitin to the C-terminus of DEDD to mimic monoubiquitinated DEDD relocated DEDD from nucleoli to the cytosol. This fusion protein also demonstrated a greater apoptosis potential than unmodified DEDD. Finally, we show that both mono- and polyubiquitination of DEDD can be achieved by the cellular inhibitor of apoptosis proteins 1 and 2 (cIAP-1/2). In addition, the cotransfection of DEDD with cIAP-1 or cIAP-2 results in the relocalization of the IAPs to the nucleoli. Our data suggest that monoubiquitination of DEDD regulates both its cytoplasmic localization and its proapoptotic potential and that IAP proteins can regulate DEDDs ubiquitination status.