Olivier Leupin

Control of the SOST Bone Enhancer by PTH Using MEF2 Transcription Factors

Expression of the osteocyte‐derived bone formation inhibitor sclerostin in adult bone requires a distant enhancer. We show that MEF2 transcription factors control this enhancer and mediate inhibition of sclerostin expression by PTH.

Does osteocytic SOST suppression mediate PTH bone anabolism

Parathyroid hormone (PTH) has bone anabolic activity when administered intermittently, affecting cells of the osteoblastic lineage at various stages, yet much remains to be learned about precisely how PTH promotes osteoblastic bone formation. Recent discoveries revealed that PTH causes transcriptional suppression of the osteocyte marker gene SOST, which encodes the potent secreted bone formation inhibitor, sclerostin. This review addresses whether osteocytes, terminally differentiated cells of the osteoblastic lineage, which are entrapped within the mineralized bone matrix, contribute to PTH-induced bone formation responses via regulation of sclerostin levels, and discusses recent evidence on how the bone anabolic responses elicited by intermittent PTH treatment or by sclerostin inhibition overlap and diverge.

Disruption of Lrp4 function by genetic deletion or pharmacological blockade increases bone mass and serum sclerostin levels

Significance Targeting WNT (Wingless-type)/β-catenin signaling has emerged as an attractive novel therapeutic approach to the treatment of bone diseases. We previously identified LRP4 (low-density lipoprotein receptor-related protein 4) as a facilitator of action of the WNT signaling antagonist SOST/sclerostin in vitro. Here, we generated bone-specific Lrp4-deficient mouse lines and anti-LRP4 antibodies selectively disrupting the Lrp4 sclerostin facilitator function. Using these novel genetic and pharmacological tools, we demonstrate that disruption of Lrp4 function induces bone gain in vivo and results in highly elevated circulating sclerostin levels. Together, these findings provide important novel insights into the role of LRP4 as a key regulator of bone homeostasis and into the mode of action of sclerostin and provide a new strategy for promoting bone gain through targeting of the WNT pathway. We identified previously in vitro LRP4 (low-density lipoprotein receptor-related protein 4) as a facilitator of the WNT (Wingless-type) antagonist sclerostin and found mutations disrupting this function to be associated with high bone mass in humans similar to patients lacking sclerostin. To further delineate the role of LRP4 in bone in vivo, we generated mice lacking Lrp4 in osteoblasts/osteocytes or osteocytes only. Lrp4 deficiency promoted progressive cancellous and cortical bone gain in both mutants, although more pronouncedly in mice deficient in osteoblast/osteocyte Lrp4, consistent with our observation in human bone that LRP4 is most strongly expressed by osteoblasts and early osteocytes. Bone gain was related primarily to increased bone formation. Interestingly, Lrp4 deficiency in bone dramatically elevated serum sclerostin levels whereas bone expression of Sost encoding for sclerostin was unaltered, indicating that osteoblastic Lrp4 retains sclerostin within bone. Moreover, we generated anti-LRP4 antibodies selectively blocking sclerostin facilitator function while leaving unperturbed LRP4–agrin interaction, which is essential for neuromuscular junction function. These antibodies increased bone formation and thus cancellous and cortical bone mass in skeletally mature rodents. Together, we demonstrate a pivotal role of LRP4 in bone homeostasis by retaining and facilitating sclerostin action locally and provide a novel avenue to bone anabolic therapy by antagonizing LRP4 sclerostin facilitator function.

TGF-β Regulates Sclerostin Expression via the ECR5 enhancer

Wnt signaling is critical for skeletal development and homeostasis. Sclerostin (Sost) has emerged as a potent inhibitor of Wnt signaling and, thereby, bone formation. Thus, strategies to reduce sclerostin expression may be used to treat osteoporosis or non-union fractures. Transforming growth factor-beta (TGF-β) elicits various effects upon the skeleton both in vitro and in vivo depending on the duration and timing of administration. In vitro and in vivo studies demonstrate that TGF-β increases osteoprogenitor differentiation but decreases matrix mineralization of committed osteoblasts. Because sclerostin decreases matrix mineralization, this study aimed to examine whether TGF-β achieves such inhibitory effects via transcriptional modulation of Sost. Using the UMR106.01 mature osteoblast cell line, we demonstrated that TGF-βTGF-β(1)-β(2)-β(3) and Activin A increase Sost transcript expression. Pharmacologic inhibition of Alk4/5/7 in vitro and in vivo decreased endogenous Sost expression, and siRNA against Alk4 and Alk5 demonstrated their requirement for endogenous Sost expression. TGF-β(1) targeted the Sost bone enhancer ECR5 and did not affect the transcriptional activity of the endogenous Sost promoter. These results indicate that TGF-β(1) controls Sost transcription in mature osteoblasts, suggesting that sclerostin may mediate the inhibitory effect of TGF-β upon osteoblast differentiation.

Age-dependent regulation of tendon crimp structure, cell length and gap width with strain.

The black-and-white patterning of tendon fascicles when visualized by light microscopy, also known as crimp, is a well-known feature of fiber-forming collagens. However, not much is known about its development, function and response to strain. The objective of this study is to investigate the interaction of tenocyte and crimp morphology as well as their changes with increasing age and acute strain. In contrast to previous studies, which used indirect measures, such as polarized light, to investigate the crimp structure, this study visualizes internal crimp structure in three dimensions without freezing, sectioning, staining or fixing the tissue, via two-photon imaging of green fluorescent protein expressing cells within mouse tail tendon fascicles. This technique further allows straining of the live tissue while visualizing changes in crimp morphology and cell shape with increasing specimen length. Combining this novel microscopy technique with computational image and data analysis revealed a complex relationship between tenocytes and the extracellular matrix that evolves with increasing age. While the reduction of crimping with strain was observed as expected, most of the crimps were gone at 0-1% strain already. Even relatively low strains of 3% led to pronounced changes in the crimp structure after relaxation, particularly in the young animals, which could not be seen with bright-field imaging. Cell length and gap width increased with strain. However, while the cells were able to return to their original length even after high strains of 6%, the gaps between the cells widened, which may imply modified cell-cell communication after overstretching.

3D Bioprinted Muscle and Tendon Tissues for Drug Development.

IntroductionIn our aging societies, there is a huge medical need fortreatments of degenerative muscle and tendon diseases, forwhichtherearecurrentlynoapprovedpharmaceuticaltherapies.Furthermore, also devastating muscle diseases that affectchildren and younger patients such as Duchenne musculardystrophyoramyothrophiclateralsclerosis(ALS)lackcurativedrug treatments. A major hurdle in new drug discovery anddevelopment is the nonexistence of normal functional humantissuesanddiseasedtissuesforcompoundscreeningandtesting.Currently,mosthigh-throughputdrugscreeningcampaignsareperformedwithtarget-centeredbiochemicalorsimplehumancell-basedassaysifthedrugtargetisknown,orwithtwo-dimensional(2D)cellculturephenotypicscreens,ifthetargetisunknown.Identifiedhitsandfurtheroptimizedcompounds(leads)arethenusually analyzed in low-throughput rodent ex vivo and/or invivo preclinicalanimalmodelsforefficacy, potency, specificityandsafety.Besidestheobviousslownessofthissteptoassessthe pharmacodynamic and physiological effects of new drugcandidatesthejumpfromhumancell-basedsystemstoanimalpreclinicalmodelsandtohumanclinicaltrialsisveryoftentoolargeandnotreliableenoughtomaster.Fortunately,recentyearshaveseenanincredibleprogressinnewapproachesgeneratingfunctional3Dhumantissuesfromnormalanddiseasedonors.Three-dimensional(3D)humanorganotypictissueculturesaregenerallymorepredictiveforinvivoeffects,becausetheymodelmuchmorein vivo tissuephysiologythanconventional2Dcellcultures.Thus,3Dtissueculturehasthepotentialtorevolutionizedrugdiscoveryanddevelopment.Theparadigmshiftfrom2Dto3Dcellcultureisalreadyshowinggreatbenefitinbasicresearchof tissues differentiation and homeostasis as well as in tissueengineeringeffortsforregenerativemedicineapplications.

Minimal mechanical load and tissue culture conditions preserve native cell phenotype and morphology in tendon-a novel ex vivo mouse explant model: MINIMAL MECHANICAL LOAD AND TENDON CULTURE CONDITIONS

Appropriate mechanical load is essential for tendon homeostasis and optimal tissue function. Due to technical challenges in achieving physiological mechanical loads in experimental tendon model systems, the research community still lacks well‐characterized models of tissue homeostasis and physiological relevance. Toward this urgent goal, we present and characterize a novel ex vivo murine tail tendon explant model. Mouse tail tendon fascicles were extracted and cultured for 6 days in a load‐deprived environment or in a custom‐designed bioreactor applying low magnitude mechanical load (intermittent cycles to 1% strain, at 1 Hz) in serum‐free tissue culture. Cells remained viable, as did collagen structure and mechanical properties in all tested conditions. Cell morphology in mechanically loaded tendon explants approximated native tendon, whereas load‐deprived tendons lost their native cell morphology. These losses were reflected in altered gene expression, with mechanical loading tending to maintain tendon specific and matrix remodeling genes phenotypic of native tissue. We conclude from this study that ex vivo load deprivation of murine tendon in minimal culture medium results in a degenerative‐like phenotype. We further conclude that onset of tissue degeneration can be suppressed by low‐magnitude mechanical loading. Thus a minimal explant culture model featuring serum‐free medium with low mechanical loads seems to provide a useful foundation for further investigations.

A Novel Microplate 3D Bioprinting Platform for the Engineering of Muscle and Tendon Tissues

Two-dimensional (2D) cell cultures do not reflect the in vivo situation, and thus it is important to develop predictive three-dimensional (3D) in vitro models with enhanced reliability and robustness for drug screening applications. Treatments against muscle-related diseases are becoming more prominent due to the growth of the aging population worldwide. In this study, we describe a novel drug screening platform with automated production of 3D musculoskeletal-tendon-like tissues. With 3D bioprinting, alternating layers of photo-polymerized gelatin-methacryloyl-based bioink and cell suspension tissue models were produced in a dumbbell shape onto novel postholder cell culture inserts in 24-well plates. Monocultures of human primary skeletal muscle cells and rat tenocytes were printed around and between the posts. The cells showed high viability in culture and good tissue differentiation, based on marker gene and protein expressions. Different printing patterns of bioink and cells were explored and calcium signaling with Fluo4-loaded cells while electrically stimulated was shown. Finally, controlled co-printing of tenocytes and myoblasts around and between the posts, respectively, was demonstrated followed by co-culture and co-differentiation. This screening platform combining 3D bioprinting with a novel microplate represents a promising tool to address musculoskeletal diseases.