The intracellular proteolytic fragment of CD44 alters CD44 function in chondrocytes

CD44 is an adhesion molecule involved in several biological functions and the primary receptor for hyaluronan (HA). In cartilage, CD44 participates in the interplay between cells and the extracellular matrix to fine tune cellular responses to cytokines and growth factors. CD44 thus can act as a mediator between individual chondrocytes and the extracellular matrix. The extracellular domain of CD44 binds to the HA/proteoglycan rich extracellular matrix, while the intracellular domain interacts with receptors like Smad1 and with the actin cytoskeleton inside the cell. Recently, we have demonstrated that chondrocyte CD44 is proteolytically cleaved from the cell surface by an endogenous, sequential, two step process. First, there is an initial extracellular cleavage of CD44 by a matrix metalloproteinase, which releases a soluble CD44 in the extracellular matrix, followed by a gamma-secretase transmembrane cleavage, which releases an intracellular tail domain of CD44.  The main focus of this study is to understand the effects of releasing a CD44 intracellular domain (ICD) that occurs via the action of the gamma-secretase cleavage, and how intracellular accumulation of such fragments might interfere with endogenous, intact, CD44 function. The first aim evaluates whether intracellular accumulation of ICD fragments can affect CD44 ability to bind to HA and retain a pericellular coat by acting as a negative competitor. Another aim was to explore the mechanisms responsible for any effects ICD over-expression might have on CD44 function. Our data suggests that intracellular CD44-ICD over-expression exerts dominant-negative effects on full length native CD44 by competing with binding to the cytoskeleton via ankyrin-3. This is the first study that looks at possible effects of releasing multiple ICD fragments via gamma-secretase cleavage in chondrocytes and as such, will provide useful information on possible significance of CD44 fragmentation in cartilage during osteoarthritis.  Ph.D

Paedomorphic Ossification in Porpoises with an Emphasis on the Vaquita (\u3ci\u3ePhocoena sinus\u3c/i\u3e)

Heterochrony, the change in timing of developmental processes, is thought to be a key process shaping the numerous limb morphologies of tetrapods. Through a delayed offset in digit development, all cetaceans (i.e., whales, dolphins, and porpoises) have evolved supernumary phalanges (hyperphalangy). Moreover, some toothed cetaceans further alter digital morphologies by delayed endochondral and perichondral ossification of individual elements. In the harbor porpoise (Phocoena phocoena), these paedomorphic patterns have created poorly ossified phalangeal elements. However, no studies have addressed this morphology in other porpoise taxa. This study documents the timing of carpal and digital epiphyseal ossification in the poorly studied vaquita (Phocoena sinus) based on radiographs (n = 18) of known-age specimens. Patterns of vaquita manus ossification were compared between other porpoise and delphinid taxa. Adult vaquitas are paedomorphic in carpal, metacarpal, and digital development as they maintain a juvenile ossification pattern relative to that of other porpoise species of equivalent ages. Vaquitas also ossify fewer carpal elements as compared to other porpoise and some delphinid cetaceans, and ossification arrests relative to that of the harbor porpoise. Vaquitas also display sexual dimorphism as females reach a greater body size and display more ossified elements in the manus relative to their paedomorphic male cohorts

OSM-induced CD44 contributes to breast cancer metastatic potential through cell detachment but not epithelial-mesenchymal transition

Background: Hormone receptor status in human breast cancer cells is a strong indicator of the aggressiveness of a tumor. Triple negative breast cancers (TNBC) are aggressive, difficult to treat, and contribute to high incidences of metastasis by possessing characteristics such as increased tumor cell migration and a large presence of the transmembrane protein, cluster of differentiation 44 (CD44) on the cell membrane. Estrogen receptor-positive (ER+) cells are less aggressive and do not migrate until undergoing an epithelial-mesenchymal transition (EMT). Methods: The relationship between EMT and CD44 during metastatic events is assessed by observing changes in EMT markers, tumor cell detachment, and migration following cytokine treatment on both parental and CD44 knockdown human breast tumor cells. Results: ER+ T47D and MCF-7 human breast cancer cells treated with OSM demonstrate increased CD44 expression and CD44 cleavage. Conversely, ER- MDA-MB-231 human breast cancer cells do not show a change in CD44 expression nor undergo EMT in the presence of OSM. In ER+ cells, knockdown expression of CD44 by shRNA did not prevent EMT but did change metastatic processes such as cellular detachment and migration. OSM-induced migration was decreased in both ER+ and ER- cells with shCD44 cells compared to control cells, while the promotion of tumor cell detachment by OSM was decreased in ER+ MCF7-shCD44 cells, as compared to control cells. Interestingly, OSM-induced detachment in ER- MDA-MB-231-shCD44 cells that normally don't detach at significant rates. Conclusion: OSM promotes both EMT and tumor cell detachment in ER+ breast cancer cells. Yet, CD44 knockdown did not affect OSM-induced EMT in these cells, while independently decreasing OSM-induced cell detachment. These results suggest that regulation of CD44 by OSM is important for at least part of the metastatic cascade in ER+ breast cancer.The following people have contributed to this work in more ways than one. Raquel Brown provided great insight and technical experience for immunofluorescent imaging. Hannah Scott provided data analysis and contributed to the growth and propagation of cells used for this study. This study was partially funded by the following grants: NIH/NCI R15CA137510, NIH/NCRR P20RR016454, NIH/NIGMS P20GM103408, NIH/NIGMS P20GM109095, Susan G. Komen Foundation KG100513, and American Cancer Society RSG-09-276-01-CSM.S

Role of bulge epidermal stem cells and TSLP signaling in psoriasis

Psoriasis is a common inflammatory skin disease involving a cross-talk between epidermal and immune cells. The role of specific epidermal stem cell populations, including hair follicle stem cells (HF-SCs) in psoriasis is not well defined. Here, we show reduced expression of c-JUN and JUNB in bulge HF-SCs in patients with scalp psoriasis. Using lineage tracing in mouse models of skin inflammation with inducible deletion of c-Jun and JunB, we found that mutant bulge HF-SCs initiate epidermal hyperplasia and skin inflammation. Mechanistically, thymic stromal lymphopoietin (TSLP) was identified in mutant cells as a paracrine factor stimulating proliferation of neighboring non-mutant epidermal cells, while mutant inter-follicular epidermal (IFE) cells are lost over time. Blocking TSLP in psoriasis-like mice reduced skin inflammation and decreased epidermal proliferation, VEGFα expression, and STAT5 activation. These findings unravel distinct roles of HF-SCs and IFE cells in inflammatory skin disease and provide novel mechanistic insights into epidermal cell interactions in inflammation.We thank Drs. M. Serrano and M. Perez-Moreno for the Gt(ROSA)26Sortrn4(ACTB-tdTomato,-EGFP)Luo/J and K15-Cre-PGR mouse lines. We are very grateful to Drs. M. Perez-Moreno, F. Real, O. Uluckan, L. Bakiri and the laboratory members of the Sibilia and Wagner groups for critical reading of the manuscript and valuable suggestions. We thank V. Bermeo, G. Medrano, S. Leceta, O. Grana, and M. Perez for their technical help and IT support. We acknowledge R. Paus laboratory members for the shipment of hair follicle samples. N.G.L. received funding from the People programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007-2013) under REA grant agreement no 608765. A.I is funded by the Institute of Health Carlos III (PI16/01430). The Wagner laboratory was funded by a grant from the Spanish Ministry of Economy and competitiveness (SAF2015-70857RE, cofounded by the European Regional Development Fund) and is supported by the ERC (ERC-AdG 2016 CSI-Fun).S

Oncostatin M Binds to Extracellular Matrix in a Bioactive Conformation: Implications for Inflammation and Metastasis

Oncostatin M (OSM) is an interleukin-6-like inflammatory cytokine reported to play a role in a number of pathological processes including cancer. Full-length OSM is expressed as a 26 kDa protein that can be proteolytically processed into 24 kDa and 22 kDa forms via removal of C-terminal peptides. In this study, we examined both the ability of OSM to bind to the extracellular matrix (ECM) and the activity of immobilized OSM on human breast carcinoma cells. OSM was observed to bind to ECM proteins collagen types I and XI, laminin, and fibronectin in a pH-dependent fashion, suggesting a role for electrostatic bonds that involves charged amino acids of both the ECM and OSM. The C-terminal extensions of 24 kDa and 26 kDa OSM, which contains six and thirteen basic amino acids, respectively, enhanced electrostatic binding to ECM at pH 6.5–7.5 when compared to 22 kDa OSM. The highest levels of OSM binding to ECM, though, were observed at acidic pH 5.5, where all forms of OSM bound to ECM proteins to a similar extent. This indicates additional electrostatic binding properties independent of the OSM C-terminal extensions. The reducing agent dithiothreitol also inhibited the binding of OSM to ECM suggesting a role for disulfide bonds in OSM immobilization. OSM immobilized to ECM was protected from cleavage by tumor-associated proteases and maintained activity following incubation at acidic pH for extended periods of time. Importantly, immobilized OSM remained biologically active and was able to induce and sustain the phosphorylation of STAT3 in T47D and ZR-75-1 human breast cancer cells over prolonged periods, as well as increase levels of STAT1 and STAT3 protein expression. Immobilized OSM also induced epithelial–mesenchymal transition-associated morphological changes in T47D cells. Taken together, these data indicate that OSM binds to ECM in a bioactive state that may have important implications for the development of chronic inflammation and tumor metastasis

Fabrication and Evaluation of Electrospun, 3D-Bioplotted, and Combination of Electrospun/3D-Bioplotted Scaffolds for Tissue Engineering Applications

Electrospun scaffolds provide a dense framework of nanofibers with pore sizes and fiber diameters that closely resemble the architecture of native extracellular matrix. However, it generates limited three-dimensional structures of relevant physiological thicknesses. 3D printing allows digitally controlled fabrication of three-dimensional single/multimaterial constructs with precisely ordered fiber and pore architecture in a single build. However, this approach generally lacks the ability to achieve submicron resolution features to mimic native tissue. The goal of this study was to fabricate and evaluate 3D printed, electrospun, and combination of 3D printed/electrospun scaffolds to mimic the native architecture of heterogeneous tissue. We assessed their ability to support viability and proliferation of human adipose derived stem cells (hASC). Cells had increased proliferation and high viability over 21 days on all scaffolds. We further tested implantation of stacked-electrospun scaffold versus combined electrospun/3D scaffold on a cadaveric pig knee model and found that stacked-electrospun scaffold easily delaminated during implantation while the combined scaffold was easier to implant. Our approach combining these two commonly used scaffold fabrication technologies allows for the creation of a scaffold with more close resemblance to heterogeneous tissue architecture, holding great potential for tissue engineering and regenerative medicine applications of osteochondral tissue and other heterogeneous tissues

Extracellular Calcium Modulates Chondrogenic and Osteogenic Differentiation of Human Adipose-Derived Stem Cells: A Novel Approach for Osteochondral Tissue Engineering Using a Single Stem Cell Source

We have previously shown that elevating extracellular calcium from a concentration of 1.8 to 8 mM accelerates and increases human adipose-derived stem cell (hASC) osteogenic differentiation and cell-mediated calcium accretion, even in the absence of any other soluble osteogenic factors in the culture medium. However, the effects of elevated calcium on hASC chondrogenic differentiation have not been reported. The goal of this study was to determine the effects of varied calcium concentrations on chondrogenic differentiation of hASC. We hypothesized that exposure to elevated extracellular calcium (8 mM concentration) in a chondrogenic differentiation medium (CDM) would inhibit chondrogenesis of hASC when compared to basal calcium (1.8 mM concentration) controls. We further hypothesized that a full osteochondral construct could be engineered by controlling local release of calcium to induce site-specific chondrogenesis and osteogenesis using only hASC as the cell source. Human ASC was cultured as micromass pellets in CDM containing transforming growth factor-β1 and bone morphogenetic protein 6 for 28 days at extracellular calcium concentrations of either 1.8 mM (basal) or 8 mM (elevated). Our findings indicated that elevated calcium induced osteogenesis and inhibited chondrogenesis in hASC. Based on these findings, stacked polylactic acid nanofibrous scaffolds containing either 0% or 20% tricalcium phosphate (TCP) nanoparticles were electrospun and tested for site-specific chondrogenesis and osteogenesis. Histological assays confirmed that human ASC differentiated locally to generate calcified tissue in layers containing 20% TCP, and cartilage in the layers with no TCP when cultured in CDM. This is the first study to report the effects of elevated calcium on chondrogenic differentiation of hASC, and to develop osteochondral nanofibrous scaffolds using a single cell source and controlled calcium release to induce site-specific differentiation. This approach holds great promise for osteochondral tissue engineering using a single cell source (hASC) and single scaffold

Keratinocyte-derived S100A9 modulates neutrophil infiltration and affects psoriasis-like skin and joint disease

[Objectives]: S100A9, an alarmin that can form calprotectin (CP) heterodimers with S100A8, is mainly produced by keratinocytes and innate immune cells. The contribution of keratinocyte-derived S100A9 to psoriasis (Ps) and psoriatic arthritis (PsA) was evaluated using mouse models, and the potential usefulness of S100A9 as a Ps/PsA biomarker was assessed in patient samples. [Methods]: Conditional S100A9 mice were crossed with DKO* mice, an established psoriasis-like mouse model based on inducible epidermal deletion of c-Jun and JunB to achieve additional epidermal deletion of S100A9 (TKO* mice). Psoriatic skin and joint disease were evaluated in DKO* and TKO* by histology, microCT, RNA and proteomic analyses. Furthermore, S100A9 expression was analysed in skin, serum and synovial fluid samples of patients with Ps and PsA. [Results]: Compared with DKO* littermates, TKO* mice displayed enhanced skin disease severity, PsA incidence and neutrophil infiltration. Altered epidermal expression of selective pro-inflammatory genes and pathways, increased epidermal phosphorylation of STAT3 and higher circulating TNFα were observed in TKO* mice. In humans, synovial S100A9 levels were higher than the respective serum levels. Importantly, patients with PsA had significantly higher serum concentrations of S100A9, CP, VEGF, IL-6 and TNFα compared with patients with only Ps, but only S100A9 and CP could efficiently discriminate healthy individuals, patients with Ps and patients with PsA. [Conclusions]: Keratinocyte-derived S100A9 plays a regulatory role in psoriatic skin and joint disease. In humans, S100A9/CP is a promising marker that could help in identifying patients with Ps at risk of developing PsA.The Wagner laboratory at the Medical University of Vienna (MUV) is supported by an ERC‐AdG 2016 CSI‐Fun‐741888, a H2020‐MSCA‐ITN 2019‐859860‐CANCERPREV grant and the MUV. GS and AR are supported by the Deutsche Forschungsgemeinschaft (DFG-FOR2886 PANDORA and the CRC1181 Checkpoints for Resolution of Inflammation). Additional funding was received by the Bundesministerium für Bildung und Forschung (BMBF; project MASCARA), the ERC-SyG 2018 (810316 4D Nanoscope), ERC-STG 2019 (853508 BARRIER BREAK) and the IMI-funded project Hippocrates. The Oxford Laboratory at the Biomolecular Research Centre at Boise State University was supported by the National Institutes of Health, NIGMS P20GM109095 and P20GM103408

Fabrication and Evaluation of Electrospun, 3D-Bioplotted, and Combination of Electrospun/3D-Bioplotted Scaffolds for Tissue Engineering Applications

Electrospun scaffolds provide a dense framework of nanofibers with pore sizes and fiber diameters that closely resemble the architecture of native extracellular matrix. However, it generates limited three-dimensional structures of relevant physiological thicknesses. 3D printing allows digitally controlled fabrication of three-dimensional single/multimaterial constructs with precisely ordered fiber and pore architecture in a single build. However, this approach generally lacks the ability to achieve submicron resolution features to mimic native tissue. The goal of this study was to fabricate and evaluate 3D printed, electrospun, and combination of 3D printed/electrospun scaffolds to mimic the native architecture of heterogeneous tissue. We assessed their ability to support viability and proliferation of human adipose derived stem cells (hASC). Cells had increased proliferation and high viability over 21 days on all scaffolds. We further tested implantation of stacked-electrospun scaffold versus combined electrospun/3D scaffold on a cadaveric pig knee model and found that stacked-electrospun scaffold easily delaminated during implantation while the combined scaffold was easier to implant. Our approach combining these two commonly used scaffold fabrication technologies allows for the creation of a scaffold with more close resemblance to heterogeneous tissue architecture, holding great potential for tissue engineering and regenerative medicine applications of osteochondral tissue and other heterogeneous tissues