Faculty Bibliography

EB1 is a microtubule plus-end tracking protein that recognizes GTP-tubulin dimers in microtubules and thus represents a unique probe to investigate the architecture of the GTP cap of growing microtubule ends. Here, we conjugated EB1 to gold nanoparticles (EB1-gold) and imaged by cryo-electron tomography its interaction with dynamic microtubules assembled in vitro from purified tubulin. EB1-gold forms comets at the ends of microtubules assembled in the presence of GTP, and interacts with the outer surface of curved and straight tubulin sheets as well as closed regions of the microtubule lattice. Microtubules assembled in the presence of GTP, different GTP analogues or cell extracts display similarly curved sheets at their growing ends, which gradually straighten as their protofilament number increases until they close into a tube. Together, our data provide unique structural information on the interaction of EB1 with growing microtubule ends. They further offer insights into the conformational changes that tubulin dimers undergo during microtubule assembly and the architecture of the GTP-cap region.

Background/Purpose: We and others have previously shown that periostin (Postn) expression is dramatically elevated in cartilage and sub-chondral bone in OA patients and surgical models of OA (medial meniscectomy and anterior crucial ligament resection or PMX, partial meniscectomy) in rodents. In vitro Postn promotes collagen and proteoglycan degradation in human chondrocytes by upregulating MMP-13 and ADAMTS4 expression. Postn controls gene expression in bone cells by interacting with avb3 integrin. However, the nature of periostin receptor(s) in chondrocytes is unknown. DDR1, a collagen-binding receptor tyrosine kinase highly expressed in chondrocytes, controls MMP-13 expression during chondrogenesis. Therefore, we hypothesized that the effect of Postn on chondrocytes is mediated by DDR1 and Postn-deficient mice (Postn^-/-) are protected from surgically-induced post-traumatic OA. Methods: (Postn^-/-) mice were purchased from Jackson Laboratory (B6;129-Postntm1Jmol/J Stock No: 009067). We subjected 3- months old littermates (Postn+/+, Postn+/- and Postn^-/-) to partial medial meniscectomy (PMX) or sham surgery, and harvested the knee joints 8 week post-surgery for histological assessment of OA progression. Human OA chondrocytes cultures were incubated in the presence or absence of the DDR1 inhibitor DDR1-IN-1 dihydrochloridein (100-500 nM) for 2 h before addition of Postn (1 mug/ml) or control vehicle to the culture medium. MMP-13 levels were determined by ELISA 24 h post stimulation. Results: We observed abundant expression of DDR1 mRNA in human chondrocytes and we found comparable levels of DDR1 in OA and normal cartilage. However, Postn expression was 3-4 times as high in OA than in normal cartilage. Pre-incubation of human cartilage explants or cultured chondrocytes with DDR1-IN-1 dihydrochloridein inhibited both constitutive and Postn-induced MMP-13 expression in a dose-dependent manner. In contrast, neutralizing antibody to alphavbeta3 integrin had no effect on Postn induction of MMP-13 expression. Co-immunoprecipitation experiments showed that Postn physically interacts with DDR1 in human chondrocytes. Furthermore, Postn^-/- mice showed reduced PMX-induced cartilage degeneration and osteophyte formation, and both Postn+/- and Postn^-/- mice had reduced subchondral bone thickening, relative to Postn+/+ mice. Conclusion: Postn^-/- mice are protected from surgically-induced post-traumatic OA, showing that Postn promotes cartilage degeneration. DDR1 mediates the stimulatory effect of Postn on MMP-13 expression. Further studies are in progress to investigate the potential of periostin as a druggable target for the treatment of OA

Recent studies have reported that chemically synthesized small activating RNA (saRNA) targeting the promoter regions of a gene can activate its expression in different cell lines. This technique can be a powerful therapeutic method for diseases caused by complete inactivation or reduced expression of specific genes. E-cadherin is a typical tumor suppressor gene. Loss of E-cadherin mediates the transition from benign lesions to invasive, metastatic cancer. In this study, several 21-nt small double-stranded RNAs (dsRNAs) targeting the promoter regions of human E-cadherin were designed and synthesized and the features of their function were investigated to study the regulatory role of dsRNA on E-cadherin expression. A new saRNA (dsEcad661) that can enhance E-cadherin expression by targeting non-coding regulatory regions in gene promoters was identified. Using dsRNA with modified base quantity and cholesterol-conjugated dsRNA, we found the antisense strand may be the guide strand of saRNA in the upregulation of E-cadherin. These findings provide several important pieces of evidence that may improve understanding of the function of saRNA and may promote its development for clinical application.

AIM/OBJECTIVE:We conducted a retrospective analysis of summary medical reports of children diagnosed with cerebral palsy (CP) to identify clinical features of antenatal onset of CP secondary to transient ischemia in utero. METHODS:The 658 brief summary reports available in the Japan Obstetric Compensation System for Cerebral Palsy were screened, and we identified cases of singleton pregnancy, delivered at gestational age ≥ 33 weeks and those with cord blood gas pH ≥ 7.20. Of the 137 cases identified, 84 were excluded for the following reasons: no evidence of ischemic brain lesion, clear post-natal causative factor of CP, presence of a congenital condition, and sentinel hypoxic event, such as uterine rupture. The demographic profiles of the 53 cases included in our analysis were compared to identify those with and without an abnormal variability in fetal heart rate. RESULTS:Between-group comparison identified an association between abnormal heart rate variability and a lower Apgar score at 1 min (2 vs 6; P < 0.001) and 5 min (5.5 vs 8; P = 0.002), and more frequent episodes of fetal movement loss (41% vs 10%; P = 0.027). An hypoxic event ≤ 1 week before delivery was more likely to be associated with abnormal heart rate variability (89%) and low Apgar score (82%), while events at > 1 week were associated with development of polyhydramnios (44%). CONCLUSION/CONCLUSIONS:In utero transient ischemic events can contribute to term or near-term CP. Careful follow-up is recommended for fetuses with a history of fetal movement loss, abnormal variability in heart rate, and polyhydramnios of unknown causes.

Mutations of claudin-19 cause severe ocular deficits that are not easily reconciled with its role in regulating the outer blood retinal barrier. ARPE-19 is a widely used culture model of the retinal pigment epithelium (RPE). ARPE-19 is unique among epithelial cell lines, because it expresses all tight junction proteins except claudin family members. ARPE-19 also loses aspects of the RPE phenotype with cell passage. This study asks whether exogenous expression of the main RPE claudins, claudin-3 and claudin-19, would restore RPE phenotype, and whether these claudins have distinct roles in RPE. An Ussing chamber was used to measure the transepithelial electrical resistance and transepithelial electrical potential. These measurements were used to estimate the permeability co-efficients of ions. The transepithelial diffusion of polyethylene glycols were used to examine the leak pathway of tight junctions. Wound-healing, quantitative RT-PCR and immunoblotting examined diverse aspects of the RPE phenotype. Over-expression of either claudin decreased the permeability of small ions and polyethylene glycol. Both claudins were slightly cation-specific, but claudin-3 was less permeable to large solutes. Claudin expression widely affected gene expression to partially restore RPE phenotype. Claudins redistributed filamentous actin from stress fibers to circumferential bands associated with tight junctions, and made wound-healing more epithelial-like. Both claudins increased the expression of genes related to RPE core functions and increased steady-state levels of phosphorylated-AKT. In conclusion, claudin-3 and claudin-19 formed general permeability barriers and affected cell morphology, proliferation, migration, AKT signaling, and gene expression. When claudins are exogenously expressed, ARPE-19 more closely model native RPE.

Embryonic germ cell migration is a vital component of the germline lifecycle. The translocation of germ cells from the place of origin to the developing somatic gonad involves several processes including passive movements with underlying tissues, transepithelial migration, cell adhesion dynamics, the establishment of environmental guidance cues and the ability to sustain directed migration. How germ cells accomplish these feats in established model organisms will be discussed in this review, with a focus on recent discoveries and themes conserved across species.

Background/Purpose: We aimed to understand the mechanism by which membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14) controls bone and cartilage homeostasis. MT1-MMP, a cell-membrane-bound proteinase with an extracellular catalytic site and a 20-amino acid cytoplasmic tail, plays a key role in postnatal bone formation. The genetic deficiency of MT1-MMP in the mouse causes dwarfism, osteopenia and severe arthritis. Deletion of MT1-MMP in bone marrow-derived mesenchymal progenitor cells (BM-MSC) recapitulates this phenotype, showing that MT1-MMP controls osteogenic differentiation in MSC. The phenotype of MT1-MMP-/- mice has been proposed to result from lack of MT1-MMP proteolytic activity. However, mounting evidence shows a variety of proteolysis-independent signaling functions of MT1-MMP. The unique tyrosine (Y573) in the MT1-MMP cytoplasmic tail is fundamental for the control of intracellular signaling. Methods: We generated a mouse with the Y573D mutation in MT1-MMP (MT1-MMP Y573D) and characterized its skeletal phenotype by histological and microCT analyses. Isolated BM-MSC were induced to differentiate into osteoblasts, chondrocytes and adipocytes, using qRT-PCR to analyze gene expression. Mouse C3H10T1/2 MSC were transfected with MT1-MMP cDNA and analyzed for Wnt signaling by luciferase reporter assays. Results: MT1-MMP Y573D mice had increased trabecular bone relative to wt littermates, marked thinning of articular cartilage with disorganized tissue architecture, clustering and cloning of chondrocytes, and pronounced decrease in bone marrow-associated and total body fat. We induced BM-MSC from wt and MT1-MMP Y573D littermates to differentiate into osteoblast and chondrocytes, and myeloid precursors into osteoclasts. The Y573D mutation dramatically increased MSC expression of osteoblast markers and strongly downregulated chondrocyte and osteoclast markers. These findings indicated that Wnt signaling is upregulated in MT1-MMP Y573D-expressing MSC. Therefore, we analyzed Wnt signaling. We transiently transfected C3H10T1/2 MSC cells in osteoblast medium with the cDNAs for wt MT1-MMP and MT1-MMP Y573D. As controls the cells were transfected with the empty vector (pcDNA) or with MT1-MMP E240A, a mutant devoid of proteolytic activity. MT1-MMP Y573D dramatically upregulated Wnt signaling relative to wt MT1-MMP and MT1-MMP E240A. Conclusion: MT1-MMP controls Wnt signaling by a mechanism independent of extracellular proteolysis and mediated by its cytoplasmic tail. MT1-MMP is a bifunctional protein, with an extracellular proteolytic activity that promotes bone formation through ECM remodeling and a cytoplasmic tail that controls osteogenesis by interacting with a key pro-osteogenic signaling pathway

A number of age-dependent degenerative diseases are caused by chronic endoplasmic reticulum (ER) stress in vital cells. In many cases, the afflicted cells suffer from ER stress since birth, but the death of irreplaceable cells occurs only late in life. Although our understanding of ER stress-induced cell death has advanced significantly, most of the known mechanisms involve pathways that signal within hours, and it remains unclear how these pathways regulate cell death that occurs only decades later. Here, I highlight the conceptual issues and suggest ways to make sense of the age-related effect of ER stress-induced cell death in degenerative diseases

What is inflammation's big idea? In this brief overview of the role of myeloid cells in inflammation, we will critically discuss what drives the initiation, amplification, and resolution of inflammation in different anatomical sites in response to different pathological stimuli. It can be argued that we have a good understanding of the basic principles that underlie myeloid cell activation and the mobilization of innate immune cells to sites of injury and infection in acute inflammation. The challenge now for inflammation biologists is to understand how resolution of this normal physiological response goes wrong in hyperacute and chronic inflammation. A better understanding of how inflammation is regulated will allow us to develop new anti-inflammatory drugs that will reduce the burden of inflammatory disease without compromising the patient's immune defenses against infectious disease. Ideally such drugs should encourage a return to homeostasis and enhance tissue repair processes.

In response to endoplasmic reticulum (ER) stress, activation of pancreatic ER kinase (PERK) coordinates an adaptive program known as the integrated stress response (ISR) by phosphorylating translation initiation factor 2α (eIF2α). Phosphorylated eIF2α is quickly dephosphorylated by the protein phosphatase 1 and growth arrest and DNA damage 34 (GADD34) complex. Data indicate that the ISR can either promote or suppress tumor development. Our previous studies showed that the ISR is activated in medulloblastoma in both human patients and animal models, and that the decreased ISR via PERK heterozygous deficiency attenuates medulloblastoma formation in Patched1 heterozygous deficient (Ptch1+/-) mice by enhancing apoptosis of pre-malignant granule cell precursors (GCPs) during cell transformation. We showed here that GADD34 heterozygous mutation moderately enhanced the ISR and noticeably increased the incidence of medulloblastoma in adult Ptch1+/- mice. Surprisingly, GADD34 homozygous mutation strongly enhanced the ISR, but significantly decreased the incidence of medulloblastoma in adult Ptch1+/- mice. Intriguingly, GADD34 homozygous mutation significantly enhanced pre-malignant GCP apoptosis in cerebellar hyperplastic lesions and reduced the lesion numbers in young Ptch1+/- mice. Nevertheless, neither GADD34 heterozygous mutation nor GADD34 homozygous mutation had a significant effect on medulloblastoma cells in adult Ptch1+/- mice. Collectively, these data imply the dual role of the ISR, promoting and inhibiting, in medulloblastoma tumorigenesis by regulating apoptosis of pre-malignant GCPs during the course of malignant transformation.