Post-translational Modification Of Collagen Research Articles

Extra-Skeletal Manifestations in Osteogenesis Imperfecta Mouse Models.

Osteogenesis imperfecta (OI) is a rare heritable connective tissue disorder of skeletal fragility with an incidence of roughly 1:15,000. Approximately 85% of the pathogenic variants responsible for OI are in the type I collagen genes, COL1A1 and COL1A2, with the remaining pathogenic OI variants spanning at least 20 additional genetic loci that often involve type I collagen post-translational modification, folding, and intracellular transport as well as matrix incorporation and mineralization. In addition to being the most abundant collagen in the body, type I collagen is an important structural and extracellular matrix signaling molecule in multiple organ systems and tissues. Thus, OI disease-causing variants result not only in skeletal fragility, decreased bone mineral density (BMD), kyphoscoliosis, and short stature, but can also result in hearing loss, dentinogenesis imperfecta, blue gray sclera, cardiopulmonary abnormalities, and muscle weakness. The extensive genetic and clinical heterogeneity in OI has necessitated the generation of multiple mouse models, the growing awareness of non-skeletal organ and tissue involvement, and OI being more broadly recognized as a type I collagenopathy.This has driven the investigation of mutation-specific skeletal and extra-skeletal manifestations and broadened the search of potential mechanistic therapeutic strategies. The purpose of this review is to outline several of the extra-skeletal manifestations that have recently been characterized through the use of genetically and phenotypically heterogeneous mouse models of osteogenesis imperfecta, demonstrating the significant potential impact of OI disease-causing variants as a collagenopathy (affecting multiple organ systems and tissues), and its implications to overall health.

Expanding the genetic and clinical spectrum of osteogenesis imperfecta: identification of novel rare pathogenic variants in type I collagen-encoding genes.

Osteogenesis imperfecta (OI) is a clinically and genetically heterogeneous skeletal disorder. The majority of affected cases are attributed to autosomal dominant pathogenic variants (PVs) found in the COL1A1 and COL1A2 genes, which encode type I collagen. However, PVs in other genes involved in collagen posttranslational modification, processing, crosslinking, osteoblast differentiation, and bone mineralization have also been associated with OI. In this study, we present the results of next-generation sequencing (NGS) analysis using a custom panel of 11 genes known to be associated with OI. This clinical study enrolled a total of 10 patients, comprising 7 male and 3 female patients from 7 families, all from the Puglia Region in South Italy, providing a detailed overview of their age, gender, family history, OI type, and non-skeletal features. The genetic analysis revealed 5 PVs in the COL1A1 gene and 2 PVs in the COL1A2 gene. Importantly, three of these PVs have not been previously reported in the literature. These include two novel heterozygous frameshift PVs in COL1A1 (c.2890_2893del and c.3887del) and one novel heterozygous missense PV in COL1A2 (c.596G>T). The identification of these previously unreported PVs expands the variant spectrum of the COL1A1 and COL1A2 genes and may have implications for accurate diagnosis, genetic counselling, and potential therapeutic interventions in affected individuals and their families.

FRI670 Osteoblast Rna Sequencing In T1d Mice Reveals Genetic Signature Of Maladaptation To Hypoxia And Compromised Collagen Synthesis

Abstract Disclosure: S. Kim: None. J. Hong: None. F. Sultana: None. E. Shelly: None. F. Korkmaz: None. J. Gimenez: None. T. Yuen: None. I. Daehn: None. J. Choi: None. M. Zaidi: None. The main feature of skeletal abnormality in type 1 diabetes (T1D) is low bone turnover with decreased osteoblastic bone formation [1, 2]. However, the mechanism of osteoblast dysfunction remains unclear. We generated T1D mice using low dose streptozotocin (STZ). T1D mice showed lower bone mass (g/cm2) at the femur (0.07 vs. 0.09*) and trends at lumbar spine (0.05 vs. 0.06, p=0.08) compared with control group at 8 weeks after inducing T1D. Osteoblasts were differentiated using bone marrow mesenchymal stem cells, and RNAseq was performed to analyze the transcriptome of osteoblasts. In gene set enrichment analysis, several pathways including immune, hypoxia, and glycolytic pathway were enriched. The osteogenic genes panel showed decreased Bglap and Col1a1 expression, together with lower expression of Bmp2 and Wnt5a, suggesting decreased bone formation might be related to the suppression of BMP and WNT signaling. Particularly, P4ha2, encoding prolyl 4-hydroxylase subunit alpha–2, which are responsible for posttranslational modification of collagen were downregulated in T1D osteoblasts (log2FC = – 1.7**). Among genes in hypoxia pathway, Egln3 encoding prolyl hydroxylase domain-containing protein 2 and Vhl encoding von Hippel-Lindau tumor suppressor, both of which stabilize HIF–α were downregulated (log2FC = – 2.8** and – 0.31*, respectively) likely for promoting HIF–α dependent gene expression in hypoxia. However, genes of the growth signaling pathway namely Pgf, Vegfa, and Igf1r were downregulated (log2FC = – 3.1**, – 0.4* and – 0.5**, respectively). Altogether, our findings suggest that maladaptation to hypoxic condition in T1D with compromised growth signaling pathway and impaired collagen synthesis might be related to osteoblasts dysfunction in T1D bone. (* p < 0.05 and ** p < 0.001) [1] Shanbhogue VV et al, JBMR. 2015[2] Rubin MR et al, JCEM. 2022 Presentation: Friday, June 16, 2023

The Role of SOX9 in IGF-II-Mediated Pulmonary Fibrosis.

Pulmonary fibrosis (PF) associated with systemic sclerosis (SSc) results in significant morbidity and mortality. We previously reported that insulin-like growth factor-II (IGF-II) is overexpressed in lung tissues and fibroblasts from SSc patients, and IGF-II fosters fibrosis by upregulating collagen type I, fibronectin, and TGFβ. We now show that IGF-II augments mRNA levels of profibrotic signaling molecules TGFβ2 (p ≤ 0.01) and TGFβ3 (p ≤ 0.05), collagen type III (p ≤ 0.01), and the collagen posttranslational modification enzymes P4HA2 (p ≤ 0.05), P3H2 (p ≤ 0.05), LOX (p = 0.065), LOXL2 (p ≤ 0.05), LOXL4 (p ≤ 0.05) in primary human lung fibroblasts. IGF-II increases protein levels of TGFβ2 (p ≤ 0.01), as well as COL3A1, P4HA2, P4Hβ, and LOXL4 (p ≤ 0.05). In contrast, IGF-II decreases mRNA levels of the collagen degradation enzymes cathepsin (CTS) K, CTSB, and CTSL and protein levels of CTSK (p ≤ 0.05). The SRY-box transcription factor 9 (SOX9) is overexpressed in SSc lung tissues at the mRNA (p ≤ 0.05) and protein (p ≤ 0.01) levels compared to healthy controls. IGF-II induces SOX9 in lung fibroblasts (p ≤ 0.05) via the IGF1R/IR hybrid receptor, and SOX9 regulates TGFβ2 (p ≤ 0.05), TGFβ3 (p ≤ 0.05), COL3A1 (p ≤ 0.01), and P4HA2 (p ≤ 0.001) downstream of IGF-II. Our results identify a novel IGF-II signaling axis and downstream targets that are regulated in a SOX9-dependent and -independent manner. Our findings provide novel insights on the role of IGF-II in promoting pulmonary fibrosis.

Significance of Type II Collagen Posttranslational Modifications: From Autoantigenesis to Improved Diagnosis and Treatment of Rheumatoid Arthritis.

Collagen type II (COL2), the main structural protein of hyaline cartilage, is considerably affected by autoimmune responses associated with the pathogenesis of rheumatoid arthritis (RA). Posttranslational modifications (PTMs) play a significant role in the formation of the COL2 molecule and supramolecular fibril organization, and thus, support COL2 function, which is crucial for normal cartilage structure and physiology. Conversely, the specific PTMs of the protein (carbamylation, glycosylation, citrullination, oxidative modifications and others) have been implicated in RA autoimmunity. The discovery of the anti-citrullinated protein response in RA, which includes anti-citrullinated COL2 reactivity, has led to the development of improved diagnostic assays and classification criteria for the disease. The induction of immunological tolerance using modified COL2 peptides has been highlighted as a potentially effective strategy for RA therapy. Therefore, the aim of this review is to summarize the recent knowledge on COL2 posttranslational modifications with relevance to RA pathophysiology, diagnosis and treatment. The significance of COL2 PTMs as a source of neo-antigens that activate immunity leading to or sustaining RA autoimmunity is discussed.

Possibilities of Plant Preparations Use for Collagen Structure and Metabolism Disturbances Correction: Modern State of Problem

The review summarizes information on possibilities of plant preparations use for collagen structure and metabolism disturbances correction. Biologically active compounds of plant origin can both stimulate and inhibit the biosynthesis of various types collagens, accelerate or slower down their catabolism, regulate the activity of enzymes involved in the collagen's metabolism. Most of the studied compounds realize their effects simultaneously by several mechanisms. Among them, the most common are the direct influence of the substance on the processes of collagen genes expression and indirect influence via TGF-beta1- pathway. In addition, a fairly common are effects on collagen synthesis by changing organism's pools of free amino acids (as the starting compounds for this protein synthesis) and by regulation of hydroxylases (performing collagen post-translational modifications and crosslinking). Besides TGF-beta1 others cytokines can also be involved in the processes of collagen metabolism regulation by compounds of plant origin. In particular, this is characteristic of triterpenes and phytoestrogens. Such a variety of methods for collagens metabolism regulation creates a wide range of possibilities for developing new preparations based on extracts or pure plant compounds able to correct connective tissue collagen structure and metabolic disorders with minimal adverse effects. Fundamentally different possibilities for the influence of plant organisms on collagens are opened with the use of genetically modified plants. Recombinant collagens allow to obtain proteins with new programmed features, making it possible to synthesize proteins with predetermined properties for medical use.

Tissue-specific collagen hydroxylation at GEP/GDP triplets mediated by P4HA2

Collagen, the most abundant organic compound of vertebrate organisms, is a supramolecular, protein-made polymer. Details of its post-translational maturation largely determine the mechanical properties of connective tissues. Its assembly requires massive, heterogeneous prolyl-4-hydroxylation (P4H), catalyzed by Prolyl-4-hydroxylases (P4HA1–3), providing thermostability to its elemental, triple helical building block. So far, there was no evidence of tissue-specific regulation of P4H, nor of a differential substrate repertoire of P4HAs. Here, the post-translational modifications of collagen extracted from bone, skin, and tendon were compared, revealing lower hydroxylation of most GEP/GDP triplets, together with fewer other residue positions along collagen a chains, in the tendon. This regulation is mostly conserved in two distant homeotherm species, mouse and chicken. The comparison of detailed P4H patterns in both species suggests a two-step mechanism of specificity. P4ha2 expression is low in tendon and its genetic invalidation in the ATDC5 cellular model of collagen assembly specifically mimics the tendon-related P4H profile. Therefore, P4HA2 has a better ability than other P4HAs to hydroxylate the corresponding residue positions. Its local expression participates in determining the P4H profile, a novel aspect of the tissue specificities of collagen assembly.

Abstract P2-21-03: Unique Collagen Peptide Signatures between Ductal Carcinoma in Situ and Invasive Breast Cancer by Mass Spectrometry Tissue Imaging

Abstract Ductal carcinoma in situ (DCIS) is characterized by inter-tumor heterogeneity that poses a therapeutic challenge due to its unpredictable recurrence and progression to invasive breast cancer (IBC). Recent publications have implicated the crucial role of the tumor microenvironment particularly stromal differences between DCIS patients who progress to IBC (progressors) and those that do not (non-progressors). However, spatial regulation of the collagen proteome has yet to be investigated in the context of disease progression in DCIS. In this study, we hypothesized that the collagen proteome was significantly altered between DCIS and IBC and that differentiating collagen peptide signatures could be related to clinical outcomes. Our initial studies investigated collagen peptide signatures in lumpectomies (n=13) annotated as DCIS, DCIS and invasive ductal carcinoma (IDC), or IDC only. We leveraged our previously published method for spatial imaging of collagen proteomics on tissue to report collagen types and collagen post-translational modifications including 40 other extracellular matrix (ECM) proteins involved in the regulation of collagen fibers. Over 1000 peaks were found to be linked to annotated pathologies or adjacent regions. Initial comparison of DCIS to IDC lesions demonstrated 63 differentially expressed peaks between these regions by unpaired, two-tailed t-test (p< 0.001). Image segmentation of the 315,541 pixels demonstrated 16 high-level hierarchical groups designating unique spatially localized ECM proteomic groups. Notably, these groups overlaid with histopathological features and pathological annotations. Next, we investigated collagen peptide signatures in a subset of DCIS samples from the Resource of Archival Breast Tissue (RAHBT) (n=37). Samples were histologically diverse within the tissue microarrays, with cribriform, micropapillary, papillary, solid, and comedo necrosis architectural patterns. In our preliminary analysis, we found two peptide peaks that could distinguish the solid subtype (n=22) from comedo necrosis (n=4) and one peak that could discriminate between the cribriform (n=8) and solid subtype (n=22) by area under the receiver operating curve (AUROC)≥0.75 and Wilson/Brown t-test (p< 0.05). Evaluated per clinical outcome, four ECM peptides showed significantly different peak intensities in progressors with IBC recurrence (n=7) compared to non-progressors (n=26) (AUROC≥0.75; Wilson/Brown t-test p< 0.05). One peptide had significantly different peak intensities between progressors with contralateral IBC recurrence and DCIS recurrence (n=5) and non-progressors (n=26) (AUROC≥0.75; Wilson/Brown t-test p< 0.05). Overall, the data suggest that unique collagen signatures in DCIS could be useful for understanding recurrence and progression to IBC. Further investigation of the spatial distribution of the collagen proteome within DCIS pathologies and relative to clinical outcome is warranted. Citation Format: Taylor S. Hulahan, Elizabeth N. Wallace, Siri H. Strand, Graham A. Colditz, E Shelley Hwang, Robert West, Laura Spruill, Jeffrey Marks, Richard R. Drake, Peggi M. Angel. Unique Collagen Peptide Signatures between Ductal Carcinoma in Situ and Invasive Breast Cancer by Mass Spectrometry Tissue Imaging [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-21-03.

The Role of RASSF1C in the Tumor Microenvironment

The tumor microenvironment (TME) plays a vital role in tumor invasion and metastasis and provides a rich environment for identifying novel therapeutic targets. The TME landscape consists of an extracellular matrix (ECM) and stromal cells. ECM is a major component of TME that mediates the interaction between cancer cells and stromal cells to promote invasion and metastasis. We have shown in published work that RASSF1C promotes cancer stem cell development, migration, and drug resistance, in part, by promoting EMT through a mechanism that involves up-regulation of the PIWIL1-piRNA axis. Consistent with this, in this study, we demonstrate that RASSF1C promotes lung cancer metastasis in vivo using an orthotopic mouse model. Interestingly, two target genes identified in a previously conducted microarray study to be up-regulated by RASSF1C in breast and non-small cell lung cancer (NSCLC) cells are prolyl 4-hydroxylase alpha-2 (P4HA2) and procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2). In cancer, P4H2A and PLOD2 are vital for collagen posttranslational modification and folding leading to the formation of a stiff ECM and induction of EMT and cancer stem cell marker gene expression, resulting in metastatic dissemination. Here, we also show that overexpression of RASSF1C up-regulates Collagen I, P4HA2, and PLOD2 in vitro. Up-regulation of P4HA2 and PLOD2 by RASSF1C was also confirmed in lung and breast cancer cells in vivo using mouse models. Further, we found that treatment of wildtype lung cancer cells or lung cancer cells overexpressing RASSF1C or PIWIL1 with piR-35127 and 46545 (both down-regulated by RASSF1C) decreased lung cancer cell invasion/migration. Taken together, our findings suggest that RASSF1C may promote lung cancer cell ECM remodeling to induce lung cancer cell stemness, invasion, and metastasis, in part, by up-regulating a previously unknown PIWIL1-P4HA2-PLOD2 pathway. Furthermore, piR-35127 and piR-46545 could potentially be important anti-metastatic tools.

Abstract B019: Discrete regulation of the collagen proteome among pathological features in DCIS and invasive breast cancer by mass spectrometry tissue imaging

Abstract Ductal carcinoma in situ (DCIS) is characterized by inter-tumor heterogeneity that poses a therapeutic challenge due to its unpredictable recurrence and progression to invasive breast cancer (IBC). In recent publications, collagen stromal differences have been reported between patients that progressed to IBC (progressors) and those that did not (non-progressors). However, details on the role spatial regulation of the collagen proteome might play in this progression have yet to be studied. Here, we investigated the pathological distribution of collagen post-translational modifications in a cohort of patients classified as progressors with ipsilateral IBC recurrence compared to non-progressors. Previously published methods for collagen proteomics by targeted tissue mass spectrometry imaging were used. The method reports collagen types and post-translational modifications within the collagen triple-helical region as well as approximately 40 other extracellular matrix (ECM) proteins involved in the regulation of collagen fibers. Initial studies investigated collagen variation in lumpectomies (n=7) with DCIS, DCIS plus invasive ductal carcinoma (IDC) or IDC only. Over 590 peptides were found to be linked to annotated pathologies. A preliminary comparison of DCIS (n=2392 spectra) to IBC (n=4696 spectra) using area under the receiver operating curve (AUROC) ≥0.85 demonstrated that 47 peptides could individually discriminate between DCIS and IBC in this limited cohort. Image segmentation of the 405,652 pixels demonstrated 11 high-level hierarchical groups designating unique spatially localized ECM proteomic groups; these groups overlaid with histopathological features and pathological annotations. A total of 87 samples from the Resource of Archival Breast Tissue (RAHBT) matched with clinical characteristics were also investigated. Cores were histologically diverse within the tissue microarrays, with cribriform, micropapillary, papillary, solid, and comedo necrosis architectural patterns. Initial results suggest certain peptides may differentiate between non-progressors and progressors with ipsilateral IBC recurrence. Our current work focuses on correlating collagen signatures to mixed pathologies and the cellular content of cores. Further investigation of the collagen proteome is warranted. Overall, the data suggest unique collagen signatures in DCIS that could be useful for understanding recurrence and progression to IBC. Citation Format: Taylor S. Hulahan, Elizabeth N. Wallace, Siri H. Strand, Robert Michael Angelo, Graham Colditz, Eun-Sil Shelley Hwang, Robert West, Laura Spruill, Jeffrey R. Marks, Richard R. Drake, Peggi M. Angel. Discrete regulation of the collagen proteome among pathological features in DCIS and invasive breast cancer by mass spectrometry tissue imaging [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr B019.

A Fe2+-dependent self-inhibited state influences the druggability of human collagen lysyl hydroxylase (LH/PLOD) enzymes.

Multifunctional human collagen lysyl hydroxylase (LH/PLOD) enzymes catalyze post-translational hydroxylation and subsequent glycosylation of collagens, enabling their maturation and supramolecular organization in the extracellular matrix (ECM). Recently, the overexpression of LH/PLODs in the tumor microenvironment results in abnormal accumulation of these collagen post-translational modifications, which has been correlated with increased metastatic progression of a wide variety of solid tumors. These observations make LH/PLODs excellent candidates for prospective treatment of aggressive cancers. The recent years have witnessed significant research efforts to facilitate drug discovery on LH/PLODs, including molecular structure characterizations and development of reliable high-throughput enzymatic assays. Using a combination of biochemistry and in silico studies, we characterized the dual role of Fe2+ as simultaneous cofactor and inhibitor of lysyl hydroxylase activity and studied the effect of a promiscuous Fe2+ chelating agent, 2,2’-bipyridil, broadly considered a lysyl hydroxylase inhibitor. We found that at low concentrations, 2,2’-bipyridil unexpectedly enhances the LH enzymatic activity by reducing the inhibitory effect of excess Fe2+. Together, our results show a fine balance between Fe2+-dependent enzymatic activity and Fe2+-induced self-inhibited states, highlighting exquisite differences between LH/PLODs and related Fe2+, 2-oxoglutarate dioxygenases and suggesting that conventional structure-based approaches may not be suited for successful inhibitor development. These insights address outstanding questions regarding druggability of LH/PLOD lysyl hydroxylase catalytic site and provide a solid ground for upcoming drug discovery and screening campaigns.

Lysyl hydroxylase 2 mediated collagen post-translational modifications and functional outcomes

Lysyl hydroxylase 2 (LH2) is a member of LH family that catalyzes the hydroxylation of lysine (Lys) residues on collagen, and this particular isozyme has been implicated in various diseases. While its function as a telopeptidyl LH is generally accepted, several fundamental questions remain unanswered: 1. Does LH2 catalyze the hydroxylation of all telopeptidyl Lys residues of collagen? 2. Is LH2 involved in the helical Lys hydroxylation? 3. What are the functional consequences when LH2 is completely absent? To answer these questions, we generated LH2-null MC3T3 cells (LH2KO), and extensively characterized the type I collagen phenotypes in comparison with controls. Cross-link analysis demonstrated that the hydroxylysine-aldehyde (Hylald)-derived cross-links were completely absent from LH2KO collagen with concomitant increases in the Lysald-derived cross-links. Mass spectrometric analysis revealed that, in LH2KO type I collagen, telopeptidyl Lys hydroxylation was completely abolished at all sites while helical Lys hydroxylation was slightly diminished in a site-specific manner. Moreover, di-glycosylated Hyl was diminished at the expense of mono-glycosylated Hyl. LH2KO collagen was highly soluble and digestible, fibril diameters were diminished, and mineralization impaired when compared to controls. Together, these data underscore the critical role of LH2-catalyzed collagen modifications in collagen stability, organization and mineralization in MC3T3 cells.

Abstract 3839: RASSF1C and tumor microenvironment

Abstract Introduction: Tumor microenvironment (TME) plays a vital role in tumor invasion and metastasis and provides a rich environment for identifying novel therapeutic targets. The TME landscape consists of extracellular matrix (ECM) and stromal cells. ECM is a major component of TME that mediates interaction between cancer cells and stromal cells to promote invasion and metastasis. We have shown in published work that RASSF1C promotes cancer stem cell development, migration, and drug resistance, in part, by promoting EMT through a mechanism that involves up-regulation of the PIWIL1-piRNA gene axis. Interestingly, one of the target genes identified by our microarray study to be upregulated by RASSF1C is P4HA2. In cancer, P4H2A is vital for collagen posttranslational modification and folding. This leads to formation of a stiff ECM and induction of cancer stem cell marker gene expression, resulting in metastatic dissemination. High expression of P4HA2 is associated with significantly lower survival of lung cancer patients. Thus, RASSF1C could promote tumor cell ECM remodeling to induce lung cancer cell stemness, invasion and metastasis by up-regulating a novel PIWIL1-P4HA2 gene axis expression. Methods: Analysis of a microarray study using H1299 cells over-expressing RASSF1C or control vector backbone identified P4HA2 as a RASSF1C gene target. RT-PCR, immunoblots, and immunofluorescence techniques were used to confirm P4HA2 gene expression using specific primers and antibodies in cells over-expressing RASSF1C or PIWIL1 along with control cells. Kaplan-Meier analysis was performed to determine the relationship of P4HA2 expression to lung adenocarcinoma patient survival using data from The Cancer Genome Atlas (TCGA). Results: Analysis of microarray data from H1299 cells over-expressing RASSF1C or control vector backbone shows that RASSF1C up-regulates P4HA2 gene expression 2-fold. The up-regulation of P4HA2 expression by RASSF1C was confirmed by RT-PCR, immunoblots, and immunofluorescence using P4HA2 gene specific primers and antibodies. Further, H1299 cells over-expressing PIWIL1 show increased P4HA2 gene expression on immunoblots. Kaplan-Meier analysis shows that high P4HA2 expression in lung adenocarcinoma patients negatively correlates with patient survival. Thus, our findings suggest the hypothesis that RASSF1C may promote lung cancer cell ECM remodeling to induce lung cancer cell stemness, invasion and metastasis, in part, by up-regulating a novel pathway involving a PIWIL1-P4HA2 gene axis. Conclusions: Investigating the role of a RASSF1C-PIWIL1-P4HA2 gene axis in ECM remodeling in lung cancer cells will provide important mechanistic information that could, in turn, lead to identification of useful biomarkers for lung cancer prognosis and targets for therapy. Citation Format: Yousef G. Amaar, Mark E. Reeves. RASSF1C and tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3839.

The Thermal Stability of the Collagen Triple Helix Is Tuned According to the Environmental Temperature.

Triple helix formation of procollagen occurs in the endoplasmic reticulum (ER) where the single-stranded α-chains of procollagen undergo extensive post-translational modifications. The modifications include prolyl 4- and 3-hydroxylations, lysyl hydroxylation, and following glycosylations. The modifications, especially prolyl 4-hydroxylation, enhance the thermal stability of the procollagen triple helix. Procollagen molecules are transported to the Golgi and secreted from the cell, after the triple helix is formed in the ER. In this study, we investigated the relationship between the thermal stability of the collagen triple helix and environmental temperature. We analyzed the number of collagen post-translational modifications and thermal melting temperature and α-chain composition of secreted type I collagen in zebrafish embryonic fibroblasts (ZF4) cultured at various temperatures (18, 23, 28, and 33 °C). The results revealed that thermal stability and other properties of collagen were almost constant when ZF4 cells were cultured below 28 °C. By contrast, at a higher temperature (33 °C), an increase in the number of post-translational modifications and a change in α-chain composition of type I collagen were observed; hence, the collagen acquired higher thermal stability. The results indicate that the thermal stability of collagen could be autonomously tuned according to the environmental temperature in poikilotherms.

Bone matrix quality in paired iliac bone biopsies from postmenopausal women treated for 12months with strontium ranelate or alendronate.

Bone quality is altered mainly by osteoporosis, which is treated with modulators of bone quality. Knowledge of their mechanisms of action is crucial to understand their effects on bone quality. The goal of our study was to compare the action of alendronate (ALN) and strontium ranelate (SrRan) on the determinants of bone quality. The investigation was performed on over 60 paired human iliac biopsies. Paired samples correspond to biopsies obtained from the same patient, one before treatment (baseline) and one after 12months of treatment, in postmenopausal women with osteoporosis. Vibrational spectroscopy (Raman and FTIRM) and nanoindentation were used to evaluate the effect of both drugs on bone quality at the ultrastructural level. Outcomes measured by vibrational spectroscopy and nanoindentation are sensitive to bone age. New bone packets are distinguished from old bone packets. Thus, the effect of bone age is distinguished from the treatment effect. Both drugs modify the mineral and organic composition in new and old bone in different fashions after 12months of administration. The new bone formed during ALN administration is characterized by an increased mineral content, carbonation and apatite crystal size/perfection compared to baseline. Post-translational modifications of collagen are observed through an increase in the hydroxyproline/proline ratio in new bone. The proteoglycan content is also increased in new bone. SrRan directly modulates bone quality through its physicochemical actions, independent of an effect on bone remodeling. Strontium cations are captured by the hydrated layer of the mineral matrix. The mineral matrix formed during SrRan administration has a lower carbonate content and crystallinity after 12months than at baseline. Strontium might create bonds (crosslinks) with collagen and noncollagenous proteins in new and old bone. The nanomechanical properties of bone were not modified with either ALN or SrRan, probably due to the short duration of administration. Our results show that ALN and SrRan have differential effects on bone quality in relation to their mechanism of action.

Effects of temperature on muscle growth and collagen deposition in zebrafish (Danio rerio)

Collagen is a major extracellular matrix component playing pivotal roles in maintaining the muscle growth and flesh texture of fish. Enhancement of collagen deposition in muscle by environmental factors serves as an effective way to improve quality of fish products. However, as a critical environmental factor in aquaculture, the effects of temperature on muscle growth and collagen metabolism in fish are largely unknown. Here, we used zebrafish (Danio rerio) as a fish model to investigate the effects of water temperature (18 ℃, 28 ℃, 34 ℃) on the growth performance, muscle structure and collagen metabolism. After the 7-week trial, the growth performance and density of muscle fiber were significantly reduced in 18 ℃ and 34 ℃ groups, compared to the control group (28 ℃). While, the collagen deposition in muscle was significantly enhanced in low temperature group (18 ℃). RNA-sequencing and qRT-PCR indicated that the transcription of collagen genes (col1a1a, col1a1b, col2a1b, etc.), genes regulating collagen synthesis (tgf-β, ctgf) and degradation (mmp2, mmp9, timp2, etc.) were down-regulated as temperature increased. Furthermore, the protein processing in endoplasmic reticulum (ER) pathway was significantly affected by temperature. The transcription of molecular chaperones of procollagen (hsp47, p4hb, sec24a, etc) in the pathway was significantly up-regulated in high temperature group. Notably, no significant difference in the hydroxyproline content was observed among groups, suggesting a temperature compensation mechanism may exist during collagen post-translational modification (hydroxylation, etc.). It was concluded that temperature exerted profound effects on muscle growth and collagen metabolism in zebrafish. Appropriate low temperature was suggested to enhance collagen deposition in fish muscle during aquaculture practice. Our data would enrich basic knowledge of regulatory mechanism of collagen metabolism in fish and help to develop strategy for producing fish products with higher quality.

Collagen fibril assembly: New approaches to unanswered questions

Collagen fibrils are essential for metazoan life. They are the largest, most abundant, and most versatile protein polymers in animals, where they occur in the extracellular matrix to form the structural basis of tissues and organs. Collagen fibrils were first observed at the turn of the 20th century. During the last 40 years, the genes that encode the family of collagens have been identified, the structure of the collagen triple helix has been solved, the many enzymes involved in the post-translational modifications of collagens have been identified, mutations in the genes encoding collagen and collagen-associated proteins have been linked to heritable disorders, and changes in collagen levels have been associated with a wide range of diseases, including cancer. Yet despite extensive research, a full understanding of how cells assemble collagen fibrils remains elusive. Here, we review current models of collagen fibril self-assembly, and how cells might exert control over the self-assembly process to define the number, length and organisation of fibrils in tissues.

Diabetoporosis: Role of nitric oxide.

Diabetoporosis, diabetic-related decreased bone quality and quantity, is one of the leading causes of osteoporotic fractures in subjects with type 2 diabetes (T2D). This is associated with lower trabecular and cortical bone quality, lower bone turnover rates, lower rates of bone healing, and abnormal posttranslational modifications of collagen. Decreased nitric oxide (NO) bioavailability has been reported within the bones of T2D patients and can be considered as one of the primary mechanisms by which diabetoporosis is manifested. NO donors increase trabecular and cortical bone quality, increase the rate of bone formation, accelerate the bone healing process, delay osteoporosis, and decrease osteoporotic fractures in T2D patients, suggesting the potential therapeutic implication of NO-based interventions. NO is produced in the osteoblast and osteoclast cells by three isoforms of NO synthase (NOS) enzymes. In this review, the roles of NO in bone remodeling in the normal and diabetic states are discussed. Also, the favorable effects of low physiological levels of NO produced by endothelial NOS (eNOS) versus detrimental effects of high pathological levels of NO produced by inducible NOS (iNOS) in diabetoporosis are summarized. Available data indicates decreased bone NO bioavailability in T2D and decreased expression of eNOS, and increased expression and activity of iNOS. NO donors can be considered novel therapeutic agents in diabetoporosis.

Transcriptomic Bioinformatic Analyses of Atria Uncover Involvement of Pathways Related to Strain and Post-translational Modification of Collagen in Increased Atrial Fibrillation Vulnerability in Intensely Exercised Mice.

Atrial Fibrillation (AF) is the most common supraventricular tachyarrhythmia that is typically associated with cardiovascular disease (CVD) and poor cardiovascular health. Paradoxically, endurance athletes are also at risk for AF. While it is well-established that persistent AF is associated with atrial fibrosis, hypertrophy and inflammation, intensely exercised mice showed similar adverse atrial changes and increased AF vulnerability, which required tumor necrosis factor (TNF) signaling, even though ventricular structure and function improved. To identify some of the molecular factors underlying the chamber-specific and TNF-dependent atrial changes induced by exercise, we performed transcriptome analyses of hearts from wild-type and TNF-knockout mice following exercise for 2 days, 2 or 6 weeks of exercise. Consistent with the central role of atrial stretch arising from elevated venous pressure in AF promotion, all 3 time points were associated with differential regulation of genes in atria linked to mechanosensing (focal adhesion kinase, integrins and cell-cell communications), extracellular matrix (ECM) and TNF pathways, with TNF appearing to play a permissive, rather than causal, role in gene changes. Importantly, mechanosensing/ECM genes were only enriched, along with tubulin- and hypertrophy-related genes after 2 days of exercise while being downregulated at 2 and 6 weeks, suggesting that early reactive strain-dependent remodeling with exercise yields to compensatory adjustments. Moreover, at the later time points, there was also downregulation of both collagen genes and genes involved in collagen turnover, a pattern mirroring aging-related fibrosis. By comparison, twofold fewer genes were differentially regulated in ventricles vs. atria, independently of TNF. Our findings reveal that exercise promotes TNF-dependent atrial transcriptome remodeling of ECM/mechanosensing pathways, consistent with increased preload and atrial stretch seen with exercise. We propose that similar preload-dependent mechanisms are responsible for atrial changes and AF in both CVD patients and athletes.

IFT20 is critical for collagen biosynthesis in craniofacial bone formation

Intraflagellar transport (IFT) is essential for assembling primary cilia required for bone formation. Disruption of IFT frequently leads to bone defects in humans. While it has been well studied about the function of IFT in osteogenic cell proliferation and differentiation, little is known about its role in collagen biosynthesis during bone formation. Here we show that IFT20, the smallest IFT protein in the IFT-B complex, is important for collagen biosynthesis in mice. Deletion of Ift20 in craniofacial osteoblasts displayed bone defects in the face. While collagen protein levels are unaffected by loss of Ift20, collagen cross-linking was significantly altered. In both Ift20:Wnt1-Cre and Ift20:Ocn-Cre mice the bones exhibit increased hydroxylysine-aldehyde deived cross-linking, and decreased lysine-aldehyde derived cross-linking. To obtain insight into the molecular mechanisms, we examined the expression levels of telopeptidyl lysyl hydroxylase 2 (LH2), and associated chaperone complexes. The results demonstrated that, while LH2 levels were unaffected by loss of Ift20, its chaperone, FKBP65, was significantly increased in Ift20:Wnt1-Cre and Ift20:Ocn-Cre mouse calvaria as well as femurs. These results suggest that IFT20 plays a pivotal role in collagen biosynthesis by regulating, in part, telopeptidyl lysine hydroxylation and cross-linking in bone. To the best of our knowledge, this is the first to demonstrate that the IFT components control collagen post-translational modifications. This provides a novel insight into the craniofacial bone defects associated with craniofacial skeletal ciliopathies.