Calreticulin accelerates corneal wound closure and mitigates fibrosis: Potential therapeutic applications.

The processes involved in regeneration of cutaneous compared to corneal tissues involve different intrinsic mechanisms. Importantly, cutaneous wounds involve healing by angiogenesis but vascularization of the cornea obscures vision. Previous studies showed that topically applied calreticulin (CALR) healed full-thickness excisional animal wounds by a tissue regenerative process markedly enhancing repair without evoking angiogenesis. In the current study, the application of CALR in a rabbit corneal injury model: (1) accelerated full wound closure by 3 days (2) accelerated delayed healing caused by corticosteroids, routinely used to prevent post-injury inflammation, by 6 days and (3) healed wounds without vascularization or fibrosis/hazing. In vitro, CALR stimulated proliferation of human corneal epithelial cells (CE) and corneal stromal cells (keratocytes) by 1.5-fold and 1.4-fold, respectively and induced migration of CE cells and keratocytes, by 72% and 85% compared to controls of 44% and 59%, respectively. As a marker of decreased fibrosis, CALR treated corneal wounds showed decreased immunostaining for α-smooth muscle actin (α-SMA) by keratocytes and following CALR treatment in vitro, decreased the levels of TGF-ß2 in human CE cells and α-SMA in keratocytes. CALR has the potential to be a novel therapeutic both, to accelerate corneal healing from various injuries and in conjunction with corticosteroids.

Calreticulin exploits TGF-ß 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 3 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, α-smooth muscle actin in human adult dermal (HDFs) and neonatal fibroblasts (HFFs) mainly via TGF-ß canonical signaling and Smad2/3 activation; RAP, an inhibitor of LRP1 blocked eCRT ECM induction. Conversely, eCRT induction of α5 and ß1 integrins was not mediated by TGF-ß signaling nor inhibited by RAP. Whereas eCRT strongly induces ECM and integrin α5 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-ß3 first via LRP1 or other receptor signaling and later induces ECM proteins via LRP1 signaling subsequently initiating TGF-ß receptor signaling for intracellular CRT (iCRT)-dependent induction of TGF-ß1 and ECM proteins. In addition, TGF-ß1 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-ß-mediated fibrosis/scarring to achieve tissue regeneration.

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.

High-level secretion of native recombinant human calreticulin in yeast.

BACKGROUND: Calreticulin (CRT) resides in the endoplasmic reticulum (ER) and functions to chaperone proteins, ensuring proper folding, and intracellular Ca(2+) homeostasis. Emerging evidence shows that CRT is a multifunctional protein with significant roles in physiological and pathological processes with presence both inside and outside of the ER, including the cell surface and extracellular space. These recent findings suggest the possible use of this ER chaperone in development of new therapeutic pharmaceuticals. Our study was focused on human CRT production in two yeast species, Saccharomyces cerevisiae and Pichia pastoris. RESULTS: Expression of a full-length human CRT precursor including its native signal sequence resulted in high-level secretion of mature recombinant protein into the culture medium by both S. cerevisiae and P. pastoris. To ensure the structural and functional quality of the yeast-derived CRTs, we compared yeast-secreted human recombinant CRT with native CRT isolated from human placenta. In ESI-MS (electrospray ionization mass spectrometry), both native and recombinant full-length CRT showed an identical molecular weight (mass) of 46,466 Da and were monomeric by non-denaturing PAGE. Moreover, limited trypsin digestion yielded identical fragment patterns of calcium-binding recombinant and native CRT suggesting that the yeast-derived CRT was correctly folded. Furthermore, both native and recombinant CRT induced cellular proliferation (MTS assay) and migration of human dermal fibroblasts (in vitro wound healing assay) with the same specific activities (peak responses at 1-10 ng/ml) indicating that the functional integrity of yeast-derived CRT was completely preserved. Simple one-step purification of CRT from shake-flask cultures resulted in highly pure recombinant CRT protein with yields reaching 75 % of total secreted protein and with production levels of 60 and 200 mg/l from S. cerevisiae and P. pastoris, respectively. Finally, cultivation of P. pastoris in a bioreactor yielded CRT secretion titer to exceed 1.5 g/l of culture medium. CONCLUSIONS: Yeasts are able to correctly process and secrete large amounts of mature recombinant human CRT equally and fully biologically active as native human CRT. This allows efficient production of high-quality CRT protein in grams per liter scale.

Exogenous calreticulin improves diabetic wound healing.

A serious consequence of diabetes mellitus is impaired wound healing, which largely resists treatment. We previously reported that topical application of calreticulin (CRT), an endoplasmic reticulum chaperone protein, markedly enhanced the rate and quality of wound healing in an experimental porcine model of cutaneous repair. Consistent with these in vivo effects, in vitro CRT induced the migration and proliferation of normal human cells critical to the wound healing process. These functions are particularly deficient in poor healing diabetic wounds. Using a genetically engineered diabetic mouse (db/db) in a full-thickness excisional wound healing model, we now show that topical application of CRT induces a statistically significant decrease in the time to complete wound closure compared with untreated wounds by 5.6 days (17.6 vs. 23.2). Quantitative analysis of the wounds shows that CRT increases the rate of reepithelialization at days 7 and 10 and increases the amount of granulation tissue at day 7 persisting to day 14. Furthermore, CRT treatment induces the regrowth of pigmented hair follicles observed on day 28. In vitro, fibroblasts isolated from diabetic compared with wild-type mouse skin and human fibroblasts cultured under hyperglycemic compared with normal glucose conditions proliferate and strongly migrate in response to CRT compared with untreated controls. The in vitro effects of CRT on these functions are consistent with CRT's potent effects on wound healing in the diabetic mouse. These studies implicate CRT as a potential powerful topical therapeutic agent for the treatment of diabetic and other chronic wounds.

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 ± 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-ß1, and integrin ß1 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: non-endoplasmic reticulum functions in physiology and disease.

Calreticulin (CRT), when localized to the endoplasmic reticulum (ER), has important functions in directing proper conformation of proteins and glycoproteins, as well as in homeostatic control of cytosolic and ER calcium levels. There is also steadily accumulating evidence for diverse roles for CRT localized outside the ER, including data suggesting important roles for CRT localized to the outer cell surface of a variety of cell types, in the cytosol, and in the extracellular matrix (ECM). Furthermore, the addition of exogenous CRT rescues numerous CRT-driven functions, such as adhesion, migration, phagocytosis, and immunoregulatory functions of CRT-null cells. Recent studies show that topically applied CRT has diverse and profound biological effects that enhance cutaneous wound healing in animal models. This evidence for extracellular bioactivities of CRT has provided new insights into this classically ER-resident protein, despite a lack of knowledge of how CRT exits from the ER to the cell surface or how it is released into the extracellular milieu. Nonetheless, it has become clear that CRT is a multicompartmental protein that regulates a wide array of cellular responses important in physiological and pathological processes, such as wound healing, the immune response, fibrosis, and cancer.-Gold, L. I., Eggleton, P., Sweetwyne, M. T., Van Duyn, L. B., Greives, M. R., Naylor, S.-M., Michalak, M., Murphy-Ullrich, J. E. Calreticulin: non-endoplamic reticulum functions in physiology and disease.

Calreticulin, a multi-process calcium-buffering chaperone of the endoplasmic reticulum.

Calreticulin is an ER (endoplasmic reticulum) luminal Ca2+-buffering chaperone. The protein is involved in regulation of intracellular Ca2+ homoeostasis and ER Ca2+ capacity. The protein impacts on store-operated Ca2+ influx and influences Ca2+-dependent transcriptional pathways during embryonic development. Calreticulin is also involved in the folding of newly synthesized proteins and glycoproteins and, together with calnexin (an integral ER membrane chaperone similar to calreticulin) and ERp57 [ER protein of 57 kDa; a PDI (protein disulfide-isomerase)-like ER-resident protein], constitutes the 'calreticulin/calnexin cycle' that is responsible for folding and quality control of newly synthesized glycoproteins. In recent years, calreticulin has been implicated to play a role in many biological systems, including functions inside and outside the ER, indicating that the protein is a multi-process molecule. Regulation of Ca2+ homoeostasis and ER Ca2+ buffering by calreticulin might be the key to explain its multi-process property.

Calreticulin enhances porcine wound repair by diverse biological effects.

Extracellular functions of the endoplasmic reticulum chaperone protein calreticulin (CRT) are emerging. Here we show novel roles for exogenous CRT in both cutaneous wound healing and diverse processes associated with repair. Compared with platelet-derived growth factor-BB-treated controls, topical application of CRT to porcine excisional wounds enhanced the rate of wound re-epithelialization. In both normal and steroid-impaired pigs, CRT increased granulation tissue formation. Immunohistochemical analyses of the wounds 5 and 10 days after injury revealed marked up-regulation of transforming growth factor-beta3 (a key regulator of wound healing), a threefold increase in macrophage influx, and an increase in the cellular proliferation of basal keratinocytes of the new epidermis and of cells of the neodermis. In vitro studies confirmed that CRT induced a greater than twofold increase in the cellular proliferation of primary human keratinocytes, fibroblasts, and microvascular endothelial cells (with 100 pg/ml, 100 ng/ml, and 1.0 pg/ml, respectively). Moreover, using a scratch plate assay, CRT maximally induced the cellular migration of keratinocytes and fibroblasts (with 10 pg/ml and 1 ng/ml, respectively). In addition, CRT induced concentration-dependent migration of keratinocytes, fibroblasts macrophages, and monocytes in chamber assays. These in vitro bioactivities provide mechanistic support for the positive biological effects of CRT observed on both the epidermis and dermis of wounds in vivo, underscoring a significant role for CRT in the repair of cutaneous wounds.

Transforming growth factor-beta, estrogen, and progesterone converge on the regulation of p27Kip1 in the normal and malignant endometrium.

Hormones and growth factors regulate endometrial cell growth. Disrupted transforming growth factor-beta (TGF-beta) signaling in primary endometrial carcinoma (ECA) cells leads to loss of TGF-beta-mediated growth inhibition, which we show herein results in lack of up-regulation of the cyclin-dependent kinase inhibitor p27(Kip1) (p27) to arrest cells in G(1) phase of the cell cycle. Conversely, in normal primary endometrial epithelial cells (EECs), TGF-beta induces a dose-dependent increase in p27 protein, with a total 3.6-fold maximal increase at 100 pmol/L TGF-beta, which was 2-fold higher in the nuclear fraction; mRNA levels were unaffected. In addition, ECA tissue lysates show a high rate of ubiquitin-mediated degradation of p27 compared with normal secretory-phase endometrial tissue (SE) such that 4% and 89% of recombinant p27 added to the lysates remains after 3 and 20 h, respectively. These results are reflected in vivo as ECA tissue lacks p27 compared with high expression of p27 in SE (P < or = 0.001). Furthermore, we show that estrogen treatment of EECs causes mitogen-activated protein kinase-driven proteasomal degradation of p27 whereas progesterone induces a marked increase in p27 in both normal EECs and ECA cells. Therefore, these data suggest that TGF-beta induces accumulation of p27 for normal growth regulation of EECs. However, in ECA, in addition to enhanced proteasomal degradation of p27, TGF-beta cannot induce p27 levels due to dysregulated TGF-beta signaling, thereby causing 17beta-estradiol-driven p27 degradation to proceed unchecked for cell cycle progression. Thus, p27 may be a central target for growth regulation of normal endometrium and in the pathogenesis of ECA.

Tumor-specific efficacy of transforming growth factor-beta RI inhibition in Eker rats.

PURPOSE: Transforming growth factor beta (TGF-beta), which generally stimulates the growth of mesenchymally derived cells but inhibits the growth of epithelial cells, has been proposed as a possible target for cancer therapy. However, concerns have been raised that whereas inhibition of TGF-beta signaling could be efficacious for lesions in which TGF-beta promotes tumor development and/or progression, systemic pharmacologic blockade of this signaling pathway could also promote the growth of epithelial lesions. EXPERIMENTAL DESIGN: We examined the effect of a TGF-beta inhibitor on mesenchymal (leiomyoma) and epithelial (renal cell carcinoma) tumors in Eker rats, which are genetically predisposed to develop these tumors with a high frequency. RESULTS: Blockade of TGF-beta signaling with the ALK5/type I TGF-beta R kinase inhibitor, SB-525334, was efficacious for uterine leiomyoma; significantly decreasing tumor incidence and multiplicity, and reducing the size of these mesenchymal tumors. However, SB-525334 was also mitogenic and antiapoptotic for epithelial cells in the kidney and exacerbated the growth of epithelial lesions present in the kidneys of these animals. CONCLUSION: Although pharmacologic inhibition of TGF-beta signaling with SB-525334 may be efficacious for mesenchymal tumors, inhibition of this signaling pathway seems to promote the development of epithelial tumors.

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 ∼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.

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.

Transforming growth factor beta signaling is disabled early in human endometrial carcinogenesis concomitant with loss of growth inhibition.

Transforming growth factor beta (TGF-beta), a potent ubiquitous endogenous inhibitor of epithelial cell growth, is secreted as a latent molecule (LTGF-beta)requiring activation for function. TGF-beta signals through the type I(TbetaRI) and type II (TbetaRII) receptors, which cooperate to phosphorylate/activate Smad2/3, the transcriptional regulators of genes that induce cell cycle arrest. That carcinomas grow in vivo suggests that they are refractory to TGF-beta. However, this has been difficult to prove because of an inability to analyze the functional status of TGF-beta in vivo as well as lack of close physiological paradigms for carcinoma cells in vitro. The current studies demonstrate that whereas primary cultures of endometrial epithelial cells derived from normal proliferative endometrium (PE; n = 10) were dose-dependently and maximally growth inhibited by 55% +/- 5.3% with 10 pM TGF-beta1, endometrial epithelial cells derived from endometrial carcinomas (ECAs; n = 10) were unresponsive (P < or = 0.0066). To determine the mechanism of TGF-beta resistance in ECAs, we analyzed the TGF-beta signaling pathway in vivo by immunohistochemistry using specific antibodies to TbetaRI and TbetaRII, Smads, and to the phosphorylated form of Smad2 (Smad2P), an indicator of cells responding to bioactive TGF-beta. Smad2P was expressed in all of the normal endometria (n = 25), and was localized to the cytoplasm and nucleus in PE, and only nuclear in the secretory endometrium. In marked contrast, Smad2P immunostaining was weak or undetectable in ECA (n = 22; P < or = 0.001) and reduced in glandular hyperplasia (n = 25) compared with normal endometrium. However, total Smad2 and Smad7 (which inhibits Smad2 activation) levels were identical in ECA and normal tissue. Consistent with loss of downstream signaling, both TbetaRI (n = 19) and TbetaRII (n = 22) protein expression were significantly reduced in ECA compared with PE (n = 11; P < or = 0.05). By in situ hybridization, the mRNA levels of TbetaRI and TbetaRII were decreased in the carcinoma cells compared with normal PE glands, suggesting that receptor down-regulation occurs at the transcriptional level. Furthermore, a somatic frameshift mutation in the polyadenine tract at the 5' end of the TbetaR-II gene was detected in two of six cases examined. Finally, the ability of explants of ECA to activate endogenous LTGF-beta was deficient compared with normal tissue (23.5% versus 7.4%). Therefore, our results suggest that loss of Smad2 signaling in ECA may be because of down-regulation of TbetaRI and TbetaRII, and/or decreased activation of LTGF-beta. Because disruption of TGF-beta signaling occurred independent of grade or degree of invasion and was evident in premalignant hyperplasia, we conclude that inactivation of TGF-beta signaling leading to escape from normal growth control occurs at an early stage in endometrial carcinogenesis, thereby defining novel molecular targets for cancer prevention.

TGF-ß activates APC through Cdh1 binding for Cks1 and Skp2 proteasomal destruction stabilizing p27kip1 for normal endometrial growth.

We previously reported that aberrant TGF-ß/Smad2/3 signaling in endometrial cancer (ECA) leads to continuous ubiquitylation of p27(kip1)(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-ß 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-ß 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-ß-mediated inhibition of proliferation. Protein synthesis was not required for TGF-ß-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-ß-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-ß signaling.

Postirradiation morphea and subcutaneous polyarteritis nodosa: case report and literature review.

OBJECTIVE: To describe a case of postirradiation morphea and subcutaneous polyarteritis nodosa occurring simultaneously in a patient and to review the literature on postirradiation autoimmune phenomenon and the potential pathogenesis of such changes. METHODS: A 75-year-old woman with breast cancer treated with chemotherapy and radiation who developed postirradiation morphea and subcutaneous polyarteritis nodosa, both inside and outside of the field of radiation, is described. Literature searches were performed on postirradiation morphea and other radiation-related inflammatory cutaneous conditions and the potential pathogenic mechanisms involved. RESULTS: Twenty-five cases of postirradiation morphea and 8 cases of postirradiation panniculitis were reported in the literature. Only 3 cases of morphea with distant vasculitis occurring in the same patient have been reported and each of these patients had features suggestive of an underlying connective tissue disease. This is the first case of morphea and subcutaneous polyarteritis nodosa occurring in the same location both inside and outside the field of radiation. CONCLUSIONS: Postirradiation morphea is an uncommon condition but is being increasingly recognized. Related phenomena following radiation include postirradiation panniculitis and now postirradiation subcutaneous polyarteritis nodosa. Radiation may be responsible for inducing some of the pathogenic changes seen in scleroderma and other autoimmune diseases. Rheumatologists should be aware of these potential complications of radiation treatment.

Gene expression changes induced by estrogen and selective estrogen receptor modulators in primary-cultured human endometrial cells: signals that distinguish the human carcinogen tamoxifen.

Tamoxifen has long been the endocrine treatment of choice for women with breast cancer and is now employed for prophylactic use in women at high risk from breast cancer. Other selective estrogen receptor modulators (SERMs), such as raloxifene, mimic some of tamoxifen's beneficial effects and, like tamoxifen, exhibit a complex mixture of organ-specific estrogen agonist and antagonistic properties. However, accompanying the positive effects of tamoxifen has been the emergence of evidence for an increased risk of endometrial cancer associated with its use. A more complete understanding of the mechanism(s) of SERM carcinogenicity and endometrial effects is therefore required. We have sought to compare and characterise the transcript profile of tamoxifen, raloxifene and the agonist estradiol in human endometrial cells. Using primary cultures of human endometria, to best emulate the in vivo responses in a manageable in vitro system, we have shown 230 significant changes in gene expression for epithelial cultures and 83 in stromal cultures, either specific to 17beta-estradiol, tamoxifen or raloxifene, or changed across more than one of the treatments. Considering the transcriptome as a whole, the endometrial responses to raloxifene or tamoxifen were more similar than either drug was to 17beta-estradiol. Treatment of endometrial cultures with tamoxifen resulted in the largest number of gene changes relative to control cultures and a high proportion of genes associated with regulation of gene transcription, cell-cycle control and signal transduction. Tamoxifen-specific changes that might point towards mechanisms for its proliferative response in the endometrium included changes in retinoblastoma and c-myc binding proteins, the APCL, dihydrofolate reductase (DHFR) and E2F1 genes and other transcription factors. Tamoxifen was also found to give rise to the highest number of gene expression changes common to those that characterise malignant endometria. It is anticipated that this study will provide leads for further and more focused investigation into SERM carcinogenicity.

Binding of polymeric IgG to fibronectin in extracellular matrices: an in vitro paradigm for immune-complex deposition.

We have previously shown a biochemical interaction between fibronectin (Fn) and polymeric immunoglobulins (Igs), that we localized to the fourth and fifth N-terminal type I repeats (4F1.5F1) in Fn and the Fc portion of IgG. Therefore, we hypothesized that Fn, as a constituent of the extracellular matrix (ECM) may directly bind circulating immune complexes (ICs) causing their deposition, thereby contributing to the pathogenesis of IC diseases. As an in vitro paradigm to test this idea, we have generated Fn-containing ECMs from varied cells in culture and demonstrated a saturable dose-dependent binding of aggregated (agg) IgG, as a prototype of ICs, as well as the binding of both heat and cold aggregated purified type I cryoglobulins (CGs) to these ECMs. No binding was observed to ECMs (Matrigel) that do not contain Fn. Characteristic of our previous findings, polymeric but not monomeric IgG bound to the acellular Fn-containing ECMs. To further demonstrate the specificity of the interaction and implicate matrix Fn in the binding of aggIgG, complete inhibition of binding of aggIgG to Fn was achieved by blocking Fn on the ECMs with anti-Fn antiserum and by preincubation of the Ig aggregates with anti-human IgG antibodies. By competing the binding interaction with fluid phase Fn and the Ig-binding site on Fn, 4F(1).5F(1), 70% inhibition was obtained. Additional experiments performed with purified CGs show that an identical dose-dependent increase in Fn binding occurred using both preformed and forming cryoprecipitates suggesting that Fn does not confer cryoprecipitation of CGs and that the specific association of Fn with cryoprecipitates probably results from their polymeric configuration. Our results support the notion that Fn, as it exits in expanding ECMs characteristic of glomerulonephropathies and rheumatoid synovial disease, specifically interacts with complexed/polymeric Igs, thereby perpetuating IC deposition and playing a role in the pathogenesis of IC diseases.

Inhibitors of SCF-Skp2/Cks1 E3 ligase block estrogen-induced growth stimulation and degradation of nuclear p27kip1: therapeutic potential for endometrial cancer.

In many human cancers, the tumor suppressor, p27(kip1) (p27), a cyclin-dependent kinase inhibitor critical to cell cycle arrest, undergoes perpetual ubiquitin-mediated proteasomal degradation by the E3 ligase complex SCF-Skp2/Cks1 and/or cytoplasmic mislocalization. Lack of nuclear p27 causes aberrant cell cycle progression, and cytoplasmic p27 mediates cell migration/metastasis. We previously showed that mitogenic 17-ß-estradiol (E2) induces degradation of p27 by the E3 ligase Skp1-Cullin1-F-Box- S phase kinase-associated protein2/cyclin dependent kinase regulatory subunit 1 in primary endometrial epithelial cells and endometrial carcinoma (ECA) cell lines, suggesting a pathogenic mechanism for type I ECA, an E2-induced cancer. The current studies show that treatment of endometrial carcinoma cells-1 (ECC-1) with small molecule inhibitors of Skp2/Cks1 E3 ligase activity (Skp2E3LIs) stabilizes p27 in the nucleus, decreases p27 in the cytoplasm, and prevents E2-induced proliferation and degradation of p27 in endometrial carcinoma cells-1 and primary ECA cells. Furthermore, Skp2E3LIs increase p27 half-life by 6 hours, inhibit cell proliferation (IC50, 14.3µM), block retinoblastoma protein (pRB) phosphorylation, induce G1 phase block, and are not cytotoxic. Similarly, using super resolution fluorescence localization microscopy and quantification, Skp2E3LIs increase p27 protein in the nucleus by 1.8-fold. In vivo, injection of Skp2E3LIs significantly increases nuclear p27 and reduces proliferation of endometrial epithelial cells by 42%-62% in ovariectomized E2-primed mice. Skp2E3LIs are specific inhibitors of proteolytic degradation that pharmacologically target the binding interaction between the E3 ligase, SCF-Skp2/Cks1, and p27 to stabilize nuclear p27 and prevent cell cycle progression. These targeted inhibitors have the potential to be an important therapeutic advance over general proteasome inhibitors for cancers characterized by SCF-Skp2/Cks1-mediated destruction of nuclear p27.

Estrogen and progesterone regulate p27kip1 levels via the ubiquitin-proteasome system: pathogenic and therapeutic implications for endometrial cancer.

The levels of proteins that control the cell cycle are regulated by ubiquitin-mediated degradation via the ubiquitin-proteasome system (UPS) by substrate-specific E3 ubiquitin ligases. The cyclin-dependent kinase inhibitor, p27kip1 (p27), that blocks the cell cycle in G1, is ubiquitylated by the E3 ligase SCF-Skp2/Cks1 for degradation by the UPS. In turn, Skp2 and Cks1 are ubiquitylated by the E3 ligase complex APC/Cdh1 for destruction thereby maintaining abundant levels of nuclear p27. We previously showed that perpetual proteasomal degradation of p27 is an early event in Type I endometrial carcinogenesis (ECA), an estrogen (E2)-induced cancer. The present studies demonstrate that E2 stimulates growth of ECA cell lines and normal primary endometrial epithelial cells (EECs) and induces MAPK-ERK1/2-dependent phosphorylation of p27 on Thr187, a prerequisite for p27 ubiquitylation by nuclear SCF-Skp2/Cks1 and subsequent degradation. In addition, E2 decreases the E3 ligase [APC]Cdh1 leaving Skp2 and Cks1 intact to cause p27 degradation. Furthermore, knocking-down Skp2 prevents E2-induced p27 degradation and growth stimulation suggesting that the pathogenesis of E2-induced ECA is dependent on Skp2-mediated degradation of p27. Conversely, progesterone (Pg) as an inhibitor of endometrial proliferation increases nuclear p27 and Cdh1 in primary EECs and ECA cells. Pg, also increases Cdh1 binding to APC to form the active E3ligase. Knocking-down Cdh1 obviates Pg-induced stabilization of p27 and growth inhibition. Notably, neither E2 nor Pg affected transcription of Cdh1, Skp2, Cks1 nor p27. These studies provide new insights into hormone regulation of cell proliferation through the UPS. The data implicates that preventing nuclear p27 degradation by blocking Skp2/Cks1-mediated degradation of p27 or increasing Cdh1 to mediate degradation of Skp2-Cks1 are potential strategies for the prevention and treatment of ECA.