Biomimetic Extracellular Matrix Nanofibers Electrospun with Calreticulin Promote Synergistic Activity for Tissue Regeneration
In recognition of the potential of calreticulin (CRT) protein in enhancing the rate and quality of wound healing in excisional animal wound models, this study was to incorporate CRT via polyblend electrospinning into polycaprolactone (PCL)/type 1 collagen (Col1) nanofibers (NFs; 334 Â± 75 nm diameter) as biomimetic extracellular matrices to provide a novel mode of delivery and protection of CRT with enhanced synergistic activities for tissue regeneration. Release kinetic studies using fluoresceinated CRT (CRT-FITC) polyblend NFs showed a burst release within 4 h reaching a plateau at 72 h, with further intervals of release upon incubation with fresh phosphate buffered saline for up to 8 weeks. By measuring fluorescence during the first 4 h of release, CRT-FITC-containing NFs were shown to protect CRT from proteolytic digestion (e.g., by subtilisin) compared to CRT-FITC in solution. CRT incorporated into NFs (CRT-NFs) also showed retention of biological activities and potency for stimulating proliferation and migration of human keratinocytes and fibroblasts. Fibroblasts seeded on CRT-NFs, after 2 days, showed increased amounts of fibronectin, TGF-Î²1, and integrin Î²1 in cell lysates by immunoblotting. Compared to NFs without CRT, CRT-NFs supported cell responses consistent with greater cell polarization and increased laminin-5 deposition of keratinocytes and a more motile phenotype of fibroblasts, as suggested by vinculin-capping F-actin fibers nonuniformly located throughout the cell body and the secretion of phosphorylated focal adhesion kinase-enriched migrasomes. Altogether, CRT electrospun into PCL/Col1 NFs retained its structural integrity and biological functions while having additional benefits of customizable loading, protection of CRT from proteolytic degradation, and sustained release of CRT from NFs, coupled with innate physicochemical cues of biomimetic PCL/Col1 NFs. Such synergistic activities have potential for healing recalcitrant wounds such as diabetic foot ulcers.
Calreticulin exploits TGF-Î² for extracellular matrix induction engineering a tissue regenerative process
Topical application of extracellular calreticulin (eCRT), an ER chaperone protein, in animal models enhances wound healing and induces tissue regeneration evidenced by epidermal appendage neogenesis and lack of scarring. In addition to chemoattraction of cells critical to the wound healing process, eCRT induces abundant neo-dermal extracellular matrix (ECM) formation by 3Â days post-wounding. The purpose of this study was to determine the mechanisms involved in eCRT induction of ECM. In vitro, eCRT strongly induces collagen I, fibronectin, elastin, Î±-smooth muscle actin in human adult dermal (HDFs) and neonatal fibroblasts (HFFs) mainly via TGF-Î² canonical signaling and Smad2/3 activation; RAP, an inhibitor of LRP1 blocked eCRT ECM induction. Conversely, eCRT induction of Î±5 and Î²1 integrins was not mediated by TGF-Î² signaling nor inhibited by RAP. Whereas eCRT strongly induces ECM and integrin Î±5 proteins in K41 wild-type mouse embryo fibroblasts (MEFs), CRT null MEFs were unresponsive. The data show that eCRT induces the synthesis and release of TGF-Î²3 first via LRP1 or other receptor signaling and later induces ECM proteins via LRP1 signaling subsequently initiating TGF-Î² receptor signaling for intracellular CRT (iCRT)-dependent induction of TGF-Î²1 and ECM proteins. In addition, TGF-Î²1 induces 2-3-fold higher level of ECM proteins than eCRT. Whereas eCRT and iCRT converge for ECM induction, we propose that eCRT attenuates TGF-Î²-mediated fibrosis/scarring to achieve tissue regeneration.
Calreticulin attenuates tgf-beta extracellular matrix for tissue regeneration [Meeting Abstract]
Background: Calreticulin (CRT), an ER chaperone, has been shown to have extracellular functions via outside-in signaling. We show that both intracellular CRT and extracellular CRT are required for the synthesis and release of the extracellular matrix (ECM) proteins, collagen, fibronectin, and elastin. Using diabetic mice, topically applied CRT for 4 days post-wounding healed full thickness excisional wounds by tissue regeneration characterized by abundant neo-dermis, epidermal appendage neogenesis, and no scarring. By day 14 post-wounding upon epidermal closure, hair follicles evaginate from the epidermis into the dermis, which show pigmentation by day 21 post-wounding. In vitro, we have shown that induction of ECM proteins by CRT in fibroblasts [to fill in the wound defect] is via TGF-beta signaling. However, as TGF-beta critically induces scarring and CRT heals by tissue regeneration, we interrogated potential mechanisms that might be involved in CRT modulation of TGF-beta-induced fibrosis/scarring using human dermal fibroblasts.
Result(s): Inhibiting protein translation with cycloheximide, we show that CRT induces a faster turn-over rate of ECM proteins than TGF-beta. On a molar basis, TGF-beta induces between 1.7-3.4 higher levels ECM proteins than CRT. Over time, the production of ECM proteins by CRT decreases earlier than by TGF-beta. The connection between TGF-beta and Wnt pathways plays a role in fibrosis by inducing ECM proteins. As a mechanism involved in fibrosis, TGF-beta was shown to downregulate DKK-1, an inhibitor of Wnt signaling. In contrast, CRT increases DKK-1 as a potential mechanism for decreasing Wnt-related betacatenin activation. Furthermore, at 3h, CRT induces miR29, known to block collagen and fibronectin transcription.
Conclusion(s): Revealing mechanisms involved in CRT attenuation of TGF-beta induced scarring should implicate new therapeutic targets for tissue regeneration of chronic wounds
The Biophysical Interaction of the Danger-Associated Molecular Pattern (DAMP) Calreticulin with the Pattern-Associated Molecular Pattern (PAMP) Lipopolysaccharide
The endoplasmic reticulum (ER) chaperone protein, calreticulin (CRT), is essential for proper glycoprotein folding and maintaining cellular calcium homeostasis. During ER stress, CRT is overexpressed as part of the unfolded protein response (UPR). In addition, CRT can be released as a damage-associated molecular pattern (DAMP) molecule that may interact with pathogen-associated molecular patterns (PAMPs) during the innate immune response. One such PAMP is lipopolysaccharide (LPS), a component of the gram-negative bacterial cell wall. In this report, we show that recombinant and native human placental CRT strongly interacts with LPS in solution, solid phase, and the surface of gram-negative and gram-positive bacteria. Furthermore, LPS induces oilgomerization of CRT with a disappearance of the monomeric form. The application of recombinant CRT (rCRT) to size exclusion and anion exchange chromatography shows an atypical heterogeneous elution profile, indicating that LPS affects the conformation and ionic charge of CRT. Interestingly, LPS bound to CRT is detected in sera of bronchiectasis patients with chronic bacterial infections. By ELISA, rCRT dose-dependently bound to solid phase LPS via the N- and C-domain globular head region of CRT and the C-domain alone. The specific interaction of CRT with LPS may be important in PAMP innate immunity.
A Complex Mechanism of Extracellular Matrix Induction by Er Chaperone Calreticulin and Tgf-beta for Tissue Regeneration [Meeting Abstract]
Hormonal and Growth Regulation of Epithelial and Stromal Cells From the Normal and Malignant Endometrium by Pigment Epithelium-Derived Factor
We discovered that pigment epithelium-derived factor (PEDF)-null mice have endometrial hyperplasia, the precursor to human type I endometrial cancer (ECA), which is etiologically linked to unopposed estrogen (E2), suggesting that this potent antiangiogenic factor might contribute to dysregulated growth and the development of type I ECA. Treatment of both ECA cell lines and primary ECA cells with recombinant PEDF dose dependently decreased cellular proliferation via an autocrine mechanism by blocking cells in G1 and G2 phases of the cell cycle. Consistent with the known opposing effects of E2 and progesterone (Pg) on endometrial proliferation, Pg increases PEDF protein synthesis and release, whereas E2 has the converse effect. Using PEDF luciferase promoter constructs containing two Pg and one E2 response elements, E2 reduced and Pg increased promoter activity due to distal response elements. Furthermore, E2 decreases and Pg increases PEDF secretion into conditioned media (CM) by both normal endometrial stromal fibroblasts (ESFs) and cancer-associated fibroblasts (CAFs), but only CM from ESFs mediated growth-inhibitory activity of primary endometrial epithelial cells (EECs). In addition, in cocultures with primary EECs, Pg-induced growth inhibition is mediated by ESFs, but not CAFs. This is consistent with reduced levels of Pg receptors on CAFs surrounding human malignant glands in vivo. Taken together, the data suggest that PEDF is a hormone-regulated negative autocrine mediator of endometrial proliferation, and that paracrine growth inhibition by soluble factors, possibly PEDF, released by ESFs in response to Pg, but not CAFs, exemplifies a tumor microenvironment that contributes to the pathogenesis of ECA.
Heightened autoantibody immune response to citrullinated calreticulin in bronchiectasis: implications for rheumatoid arthritis
Calreticulin (CRT) and citrullinated (citCRT) are implicated in rheumatoid arthritis (RA) pathology. citCRT binds to RA shared epitopes (SE) on HLA-DR molecules with high affinity and triggers pro-inflammatory events in adjacent cells. The aim of the study was to detect the presence of citCRT prior to developing RA and evaluate if citCT is a target for autoantibodies in RA cohorts with and without lung disease. Antibodies were assessed by ELISA against native CRT, citCRT and general protein citrullination, in sera from 50 RA patients without lung disease, 122 bronchiectasis (BR) patients, 52 bronchiectasis patients with RA (BRRA), 87 asthma patients and 77 healthy controls (HC). Serum citCRT was detected by immunoblotting and mass spectrometry. Genomic DNA was genotyped for HLA-DRB1 alleles. Patients were assessed for DAS28, rheumatoid factor, and anti-cyclic citrullinated peptide antibodies. Extracellular citCRT was detected in BR patients sera prior to them developing RA. A citCRT SE binding peptide GEWKPR261citQIDNPDYK was identified. Anti-CRT antibodies were observed in 18% of BR patients with or without RA. Anti-citCRT antibodies were observed in approximately 35% of BR or RA patients, increasing to 58% in BRRA patients. In the RA alone patients 7/20 (35%) who were negative for RF and anti-CCP were anti-CRT antibody positive and had higher DAS28 scores than triple negative RA alone patients. Three of the four BR patients who developed RA over 18 months were anti-citCRT+ve SE+ve. The detection of citCRT in BR and development of anti-citCRT in BR patients suggests citCRT antigens are early targets of antigenicity in these patients, especially in SE+ve patients prior to the onset of RA.
Intracellular and extracellular calreticulin are required for extracellular matrix and integrin induction by a complex mechanism essential for tissue regeneration and wound healing. [Meeting Abstract]
TGF-beta activates APC through Cdh1 binding for Cks1 and Skp2 proteasomal destruction stabilizing p27kip1 for normal endometrial growth
We previously reported that aberrant TGF-beta/Smad2/3 signaling in endometrial cancer (ECA) leads to continuous ubiquitylation of p27kip 1 (p27) by the E3 ligase SCF-Skp2/Cks1 causing its degradation as a putative mechanism involved in the pathogenesis of this cancer. In contrast, normal intact TGF-beta signaling prevents degradation of nuclear p27 by SCF-Skp2/Cks1 thereby accumulating p27 to block Cdk2 for growth arrest. Here we show that in ECA cell lines and normal primary endometrial epithelial cells, TGF-beta increases Cdh1 and its binding to APC/C to form the E3 ligase complex that ubiquitylates Cks1 and Skp2 prompting their proteasomal degradation and thus, leaving p27 intact. Knocking-down Cdh1 in ECA cell lines increased Skp2/Cks1 E3 ligase activity, completely diminished nuclear and cytoplasmic p27, and obviated TGF-beta-mediated inhibition of proliferation. Protein synthesis was not required for TGF-beta-induced increase in nuclear p27 and decrease in Cks1 and Skp2. Moreover, half-lives of Cks1 and Skp2 were extended in the Cdh1-depleted cells. These results suggest that the levels of p27, Skp2 and Cks1 are strongly or solely regulated by proteasomal degradation. Finally, an inverse relationship of low p27 and high Cks1 in the nucleus was shown in patients in normal proliferative endometrium and grade I-III ECAs whereas differentiated secretory endometrium showed the reverse. These studies implicate Cdh1 as the master regulator of TGF-beta-induced preservation of p27 tumor suppressor activity. Thus, Cdh1 is a potential therapeutic target for ECA and other human cancers showing an inverse relationship between Cks1/Skp2 and p27 and/or dysregulated TGF-beta signaling.
Unfolding the complexities of ER chaperones in health and disease: report on the 11th international calreticulin workshop
The 11th International Calreticulin workshop was held May 15-18, 2015 at New York University School of Medicine-Langone Medical Center, New York. The meeting highlighted many of the new discoveries in the past 2 years involving the important role of molecular chaperones in physiological and pathological processes. Crucial to the understanding of these disease processes was the role of chaperones in maintaining quality control of protein processing in the endoplasmic reticulum, the importance of Ca(2) regulation acting through its action in stress-related diseases, and the trafficking of glycoproteins to the cell surface. Central to maintaining healthy cell physiology is the correct ER-associated protein degradation of specific misfolded proteins. Information on different mechanisms involved in the degradation of misfolded proteins was revealed. This was a landmark meeting for the chaperone field in terms of new insights into their roles in physiology. These insights included the unfolded protein response, innate/adaptive immunity, tissue repair, the functions of calreticulin/chaperones from the cell surface, and extracellular environment. Diseases included neurodegenerative disorders, prion disease, autoimmunity, fibrosis-related disease, the host immune response to cancer, and hematologic diseases associated with calreticulin mutations. The 12th calreticulin workshop is planned for the spring of 2017 in Delphi, Greece.