Biology subjects

Diederichs, S.

Publications and source records attributed to Diederichs, S..

8 recordsLinked to original sources

bioRxiv2025

Dichotomous SMAD2/3 regulation and selective anti-hypertrophic activity of heparin during in vitro chondrogenesis of mesenchymal stromal cells

BackgroundEndochondral instead of chondral differentiation hinders mesenchymal stromal cell (MSC) application for clinical cartilage regeneration. We previously showed that heparin-polyethylene glycol (PEG) hydrogels loaded with transforming growth factor beta (TGF-{beta}) instructed stable chondral MSC development in vivo. We here assessed this approach in vitro, utilizing heparin-PEG hydrogels or soluble heparin supplementation of chondrogenic medium. MethodsHuman MSCs were cultured in heparin-PEG hydrogels (22.4 mg/mL crosslinked heparin, 120ng TGF-{beta}1) or as hydrogel-free pellet cultures treated with soluble heparin (0, 10, 100, 700 g/mL) in TGF-{beta}1-containing (10 ng/mL) chondrogenic medium. Chondral and endochondral signaling (1-3 h, 4 weeks) and cartilage matrix formation (4 weeks) were analyzed using Western blot, histology, qPCR, ELISA, and enzyme activity. ResultsUnlike in vivo, human MSCs differentiated in heparin-PEG hydrogels into type X collagen and alkaline phosphatase-positive hypertrophic chondrocytes in vitro. Interestingly, treatment with soluble heparin (10-700 {micro}g/mL) revealed reduced TGF-{beta}-small mother against decapentaplegic (SMAD)3 but not SMAD2 activation at unaffected type II collagen and proteoglycan/DNA levels. We propose that the stimulation of the insulin-AKT pathway by heparin aided in maintaining SMAD2 activation which apparently plays a more prominent role than SMAD3 for MSC chondrogenesis. Heparin treatment inhibited the pro-hypertrophic WNT/{beta}-catenin pathway in vitro but insufficiently silenced TGF-{beta}-SMAD1/5/9 activation and unfortunately reduced anti-hypertrophic prostaglandin E2 (PGE2) levels. Ultimately, treatment with 10 {micro}g/mL heparin reduced the upregulation of several hypertrophy markers (MEF2C, IHH, IBSP mRNAs, alkaline phosphatase activity) below control levels, but type X collagen remained unresponsive. Thus, soluble heparin treatment was similarly selective and effective as previous anti-hypertrophic interventions (parathyroid-hormone related protein (PTHrP)-pulses, wingless-int (WNT)-inhibition), while offering technical simplicity, reduced cost, and solvent-free formulation. ConclusionsTaken together, heparin-TGF-{beta} showed a novel dichotomous SMAD2/3 inhibition at maintained chondrogenic power and context-dependent lineage-instructive properties: permitting endochondral differentiation in vitro but chondral development in vivo. Thus, environmental contributions are mandatory to allow heparin-PEG-guided chondral versus endochondral lineage commitment of MSCs in vivo, potentially involving SMAD1/5/9 suppressors and PGE2 sources. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/673657v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@140313aorg.highwire.dtl.DTLVardef@160049dorg.highwire.dtl.DTLVardef@cee22corg.highwire.dtl.DTLVardef@62e961_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

cell biology↗

bioRxiv2024

The neuropeptide aCGRP impairs the chondrocyte response to mechanical load

ObjectiveNovel targets for osteoarthritis therapy are urgently needed, and sensory nerve fibres and their neuropeptides are increasingly recognised for their contribution to structural aspects of joint pathology. The nociceptive sensory neuropeptide alpha calcitonin gene-related peptide (CGRP) was previously detected in synovial fluid and serum of osteoarthritis patients and was also described as trophic factor for chondrocytes, affecting ECM organisation and biomechanical properties. Here, we investigated the potential of CGRP to alter the chondrocyte mechanoresponse, and thus to affect the resilience of cartilage towards mechanical loading. MethodsTissue-engineered neocartilage based on human articular chondrocytes was treated with 1{micro}M CGRP for 24 hours and subjected to an anabolic loading protocol (intermittent dynamic compression, 1Hz, 25%) for the last 3 hours before analysing its molecular mechano-response. ResultsMechanotransduction was largely unaltered by CGRP as demonstrated by ERK activation and stimulation of mechano-regulated gene expression, yet load-stimulated glycosaminoglycan synthesis was disturbed by CGRP. Presence of CGRP did not affect stimulation of WNT5A expression by loading, but decreased DKK3 expression under loading. Importantly, WNT inhibition prevented the negative effect of CGRP on load-stimulated glycosaminoglycan synthesis. ConclusionIdentifying WNT5A as a novel mechano-response gene, we reveal that CGRP can block the load-stimulated proteoglycan production of human chondrocytes in the presence of WNT pathway activity. Thus, our data propose a novel, negative role for the pain-mediator CGRP in the cartilage loading response, compromising its resilience to loading. Overall, our study implicates CGRP as a potential target for osteoarthritis treatment, but also in patient stratification.

cell biology↗

bioRxiv2024

Load activated FGFR and beta1 integrins target distinct chondrocyte mechano-response genes

In response to mechanical stimuli, chondrocytes adapt their transcriptional activity, thereby shaping the cellular mechano-response; however, it remains unclear whether the activation of cell surface receptors during mechanical loading converge in the activation of the same mechano-response genes, or whether pathway-specific genes can be defined. We aimed to determine whether load-activated FGF/FGFR signalling and {beta}1 integrin jointly activate ERK and control the same or distinct subsets of mechano-regulated genes. To this end, tissue-engineered neocartilage was generated from murine costal chondrocytes or human articular chondrocytes and subjected to dynamic unconfined compression with or without FGFR inhibition. To assess the role of {beta}1 integrins, neocartilage was generated from embryonic {beta}1 integrin-deficient or wild type costal chondrocytes. Load-activated FGFR signalling drove ERK activation in murine chondrocytes, and partially also in human chondrocytes, and mechano-response genes could be classified according to their regulation: Fosl1, Itga5, Ngf and Timp1 were regulated by load-activated FGFR depending on the developmental stage, whereas {beta}1 integrins controlled Inhba expression. In human chondrocytes, load-activated FGFR controlled expression of BMP2, PTGS2 and DUSP5, but not FOSB. We show here that the chondrocyte loading response is coordinated by concurrent activation of multiple receptors, and identified for the first time distinct target genes of activated receptors. These insights open up the opportunity to pharmacologically shape the mechano-response of chondrocytes in future studies with promising implications for the management of osteoarthritis and the development of novel therapeutic strategies.

cell biology↗

bioRxiv2024

An atlas of RNA-dependent proteins in cell division reveals the riboregulation of mitotic protein-protein interactions

Ribonucleoprotein complexes are dynamic assemblies of RNA with RNA-binding proteins (RBPs), which can modulate the fate of the RNA molecules from transcription to degradation. Vice versa, RNA can regulate the interactions and functions of the associated proteins. Dysregulation of RBPs is linked to diseases such as cancer and neurological disorders. RNA and RBPs are present in mitotic structures like the centrosomes and spindle microtubules, but their influence on mitotic spindle integrity remains unknown. Thus, we applied the R-DeeP strategy for the proteome-wide identification of RNA-dependent proteins and complexes to cells synchronized in mitosis versus interphase. The resulting atlas of RNA-dependent proteins in cell division can be accessed through the R-DeeP 3.0 database (R-DeeP3.dkfz.de). It revealed key mitotic factors as RNA-dependent such as AURKA, KIFC1 and TPX2 that were linked to RNA despite their lack of canonical RNA-binding domains. KIFC1 was identified as a new interaction partner and phosphorylation substrate of AURKA at S349 and T359. In addition, KIFC1 interacted with both, AURKA and TPX2, in an RNA-dependent manner. Our data suggest a riboregulation of mitotic protein-protein interactions during spindle assembly, offering new perspectives on the control of cell division processes by RNA-protein complexes. HighlightsO_LIDifferential R-DeeP screens in mitosis and interphase are provided as a resource in a user-friendly database at R-DeeP3.dkfz.de C_LIO_LIAn atlas of RNA-dependent proteins in cell division identifies a substantial number of unconventional RNA-binding proteins among mitotic factors C_LIO_LIInvestigation of protein-protein interactions reveals KIFC1 as a new AURKA and TPX2 interaction partner during spindle assembly C_LIO_LIKIFC1, AURKA and TPX2 interact with each other in an RNA-dependent manner and directly bind to RNA C_LIO_LIAURKA phosphorylates KIFC1 at residues S349 and T359 C_LI

cell biology↗

bioRxiv2024

Mesenchymal stromal cell chondrogenesis under ALK1/2/3-specific BMP inhibition: A revision of the prohypertrophic signalling network concept

BackgroundIn vitro chondrogenesis of mesenchymal stromal cells (MSCs) driven by the essential chondro-inducer transforming growth factor (TGF)-{beta} is instable and yields undesired hypertrophic cartilage predisposed to bone formation in vivo. TGF-{beta} can non-canonically activate bone morphogenetic protein-associated ALK1/2/3 receptors. These have been accused of driving hypertrophic MSC misdifferentiation, but data remained conflicting. We here tested the antihypertrophic capacity of two highly specific ALK1/2/3 inhibitors - compound A (CompA) and LDN 212854 (LDN21) - in order to reveal potential prohypertrophic contributions of these BMP/non-canonical TGF-{beta} receptors during MSC in vitro chondrogenesis. MethodsStandard chondrogenic pellet cultures of human bone marrow-derived MSCs were treated with TGF-{beta} and CompA (500 nM) or LDN21 (500 nM). Daily 6-hour pulses of parathyroid hormone-related peptide (PTHrP[1-34], 2.5 nM, from day 7) served as potent antihypertrophic control treatment. Day 28 samples were subcutaneously implanted into immunodeficient mice. ResultsAll groups underwent strong chondrogenesis, but GAG/DNA deposition and ACAN expression were slightly but significantly reduced by ALK inhibition compared to solvent controls along with a mild decrease of the hypertrophy markers IHH-, SPP1-mRNA, and Alkaline phosphatase (ALP) activity. When corrected for the degree of chondrogenesis (COL2A1 expression), only pulsed PTHrP but not ALK1/2/3 inhibition qualified as antihypertrophic treatment. In vivo, all subcutaneous cartilaginous implants mineralized within 8 weeks, but PTHrP pretreated samples formed less bone and attracted significantly less haematopoietic marrow than ALK1/2/3 inhibitor groups. ConclusionsOverall, our data show that BMP-ALK1/2/3 inhibition cannot program mesenchymal stromal cells toward stable chondrogenesis. BMP-ALK1/2/3 signalling is no driver of hypertrophic MSC misdifferentiation and BMP receptor induction is not an adverse prohypertrophic side effect of TGF-{beta} that leads to endochondral MSC misdifferentiation. Instead, the prohypertrophic network comprises misregulated PTHrP/hedgehog signalling and WNT activity, and a potential contribution of TGF-{beta}-ALK4/5-mediated SMAD1/5/9 signalling should be further investigated to decide about its postulated prohypertrophic activity. This will help to successfully engineer cartilage replacement tissues from MSCs in vitro and translate these into clinical cartilage regenerative therapies.

cell biology↗

bioRxiv2024

Cause and chondroprotective effects of prostaglandin E2 secretion during mesenchymal stromal cell chondrogenesis

Mesenchymal stromal cells (MSCs) that are promising for cartilage tissue engineering secrete high amounts of prostaglandin E2 (PGE2), an immunoactive mediator involved in endochondral bone development. This study aimed to identify drivers of PGE2 and its role in the inadvertent MSC misdifferentiation into hypertrophic chondrocytes. PGE2 release which rose in the first three weeks of MSC chondrogenesis was jointly stimulated by endogenous BMP, WNT, and hedgehog activity that supported the exogenous stimulation by TGF-{beta}1 and insulin, and overcame the PGE2 inhibition by dexamethasone. Experiments with PGE2 treatment or the inhibitor celecoxib or specific receptor antagonists demonstrated that although driven by prohypertrophic signals, PGE2 exerted broad autocrine antihypertrophic effects. This chondroprotective effect makes PGE2 not only a promising option for future combinatorial approaches to direct MSC tissue engineering approaches into chondral instead of endochondral development, but could potentially have implications for the use of COX-2-selective inhibitors in osteoarthritis pain management.

cell biology↗

bioRxiv2023

Refining the pool of RNA-binding domains advances the classification and prediction of RNA-binding proteins

Key PointsO_LIComprehensive analysis of RNA-related protein domains and families enriched in RNA-binding proteins (RBPs) C_LIO_LIPan-species prediction of new RBPs, and prediction and validation of new RNA-binding domains C_LIO_LIOnline resource with complete dataset including high-confidence human RBPs according to a new scoring system C_LI From transcription to decay, RNA-binding proteins (RBPs) influence RNA metabolism. Using the RBP2GO database that combines proteome-wide RBP screens from 13 species, we investigated the RNA-binding features of 176896 proteins. By compiling published lists of RNA-binding domains (RBDs) and RNA-related protein family (Rfam) IDs with lists from the InterPro database, we analyzed the distribution of the RBDs and Rfam IDs in RBPs and non-RBPs to select RBDs and Rfam IDs that were enriched in RBPs. We also explored proteins for their content in intrinsically disordered regions (IDRs) and low complexity regions (LCRs). We found a strong positive correlation between IDRs and RBDs and a co-occurrence of specific LCRs. Our bioinformatic analysis indicated that RBDs/Rfam IDs were strong indicators of the RNA-binding potential of proteins and helped predicting new RBP candidates, especially in less investigated species. By further analyzing RBPs without RBD, we predicted new RBDs that were validated by RNA-bound peptides. Finally, we created the RBP2GO composite score by combining the RBP2GO score with new quality factors linked to RBDs and Rfam IDs. Based on the RBP2GO composite score, we compiled a list of 2018 high-confidence human RBPs. The knowledge collected here was integrated into the RBP2GO database at https://RBP2GO-2-Beta.dkfz.de. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/553134v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@ab464aorg.highwire.dtl.DTLVardef@93a49dorg.highwire.dtl.DTLVardef@11c771borg.highwire.dtl.DTLVardef@1cfb737_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGRAPHICAL ABSTRACTC_FLOATNO C_FIG

bioinformatics↗

bioRxiv2023

Low level of antioxidant capacity biomarkers but not target overexpression predicts vulnerability to ROS-inducing drugs

Despite a strong rationale for why cancer cells are susceptible to redox-targeting drugs, such drugs often face tumor resistance or dose-limiting toxicity in preclinical and clinical studies. An important reason is the lack of specific biomarkers to better select susceptible cancer entities and stratify patients. Using a large panel of lung cancer cell lines, we identified a set of "antioxidant-capacity" biomarkers (ACB), which were tightly repressed, partly by STAT3 and STAT5A/B in sensitive cells, rendering them susceptible to multiple redox-targeting and ferroptosis-inducing drugs. Contrary to expectation, constitutively low ACB expression was not associated with an increased steady state level of reactive oxygen species (ROS) but a high level of nitric oxide, which is required to sustain high replication rates. Using ACBs, we identified cancer entities with a high percentage of patients with favorable ACB expression pattern, making it likely that more responders to ROS-inducing drugs could be stratified for clinical trials.

cancer biology↗