Matrix Biology

Amphiregulin (AREG), a growth factor prominently expressed by immune cells, has emerged as an important mediator of tissue healing. However, its therapeutic potential and immunomodulatory effects remain elusive. Here, we engineered an optimized AREG (eAREG) with enhanced signaling and show that it promotes robust skin repair and muscle regeneration in murine models. Beyond its growth factor function, eAREG acts on tissue-resident non-immune cells to suppress inflammation-induced chemokine programs, thereby limiting the recruitment of pro-inflammatory immune cells. We further demonstrate that eAREG constrains chromatin accessibility at regulatory regions of key chemokine genes, revealing an epigenetic mechanism of immune regulation operating within non-immune tissue compartments. Importantly, the regenerative and immunomodulatory effects of eAREG are preserved in diabetic mice with elevated inflammation and impaired healing. Together, these findings identify eAREG as a dual-function regenerative biologic and establish a design principle for regenerative therapies that integrate morphogenic signaling with immunomodulation mediated by tissue-resident cells.

Pulmonary fibrosis is characterized by extracellular matrix (ECM) deposition driven by fibroblast-to-myofibroblast transition (FMT) and by an altered proteolytic balance. While the roles of several cysteine proteases have been documented, the specific contribution of cathepsin V (CatV) and legumain (LGMN) remains poorly explored. LGMN, CatV and their dual inhibitor cystatin M/E (CysM/E) are significantly increased in lung specimens and bronchoalveolar lavage fluids from patients with idiopathic pulmonary fibrosis. TGF-{beta}1 triggered CysM/E expression and LGMN transcription, intracellular maturation, enzymatic activity, and pro-LGMN secretion via the Smad-3 pathway, whereas CatV was downregulated in human lung fibroblasts (CCD-19Lu and primary HPF cells) undergoing myodifferentiation. Genetic silencing of LGMN or CatV, and pharmacological inhibition of LGMN, led to accumulation of fibronectin and elastin, implying that both proteases contribute to ECM remodeling. LGMN cleaved fibronectin, while CatV predominantly regulated elastin levels. Conversely, broad-spectrum inhibitor cystatin C (hCC) markedly reduced elastin and fibronectin degradation, whereas CysM/E exerted a weaker effect, mainly on elastin turnover. LGMN inhibition transiently delayed fibroblast wound closure, establishing a functional role in tissue repair through fibronectin remodeling. Neither LGMN nor CatV influenced -SMA expression, distinguishing them from CatB, which participates in FMT. Altogether, LGMN was identified as an effector of matrix remodeling rather than myodifferentiation, acting in concert with CatV. Within the proteolytic network governing fibrosis progression, the present findings identify a cystatin-regulated LGMN/CatV partnership, participating in ECM turnover and cell migration. Present results also provide new perspectives on potential therapeutic protease-based strategies targeting ECM turnover underlying lung fibrosis.

Heparan sulfate proteoglycans (HSPGs) are essential cell surface and extracellular matrix glycoconjugates that mediate diverse biological processes through interactions between their heparan sulfate (HS) chains and extracellular ligands. While HS sulfation patterning is known to dictate ligand specificity, how cells control HS assembly to regulate these interactions remains incompletely understood. To systematically identify genetic modifiers of HS-protein interactions, we performed genome-wide CRISPR activation (CRISPRa) screens in HEK293T cells using binding of antithrombin (AT), which selectively recognizes 3-O-sulfated HS motifs, or the N-sulfation-specific antibody 10E4 as functional readouts. Strikingly, the screens revealed proteoglycan core proteins as key modulators of HS function. In particular, syndecan-1 (SDC1) emerged as a preferential enhancer of AT binding compared to other syndecan family members. Targeted upregulation of syndecan family members increased total HS levels, but only SDC1 enhanced AT binding. Structural and enzymatic analyses demonstrated that SDC1-associated HS chains contain elevated 6-O-sulfation and serve as superior substrates for 3-O-sulfotransferases relative to SDC2-associated HS chains. Additionally, SDC1 exhibited slower cell surface recovery, which was blocked by cycloheximide treatment, consistent with extended trafficking and biosynthetic processing. Overall, these findings indicate that proteoglycan core protein identity influences HS sulfation patterning and ligand-binding specificity and trafficking kinetics may contribute to core protein-dependent regulation of HS modification.

The commensal microbiota actively shapes skin homeostasis by regulating immune responses and barrier function, yet interactions with the dermis and dermal fibroblasts remain poorly defined. Here, we show that the commensal microbiota regulates dermal collagen homeostasis through epidermal-mediated signaling. Transcriptomic analysis revealed enrichment of extracellular matrix (ECM)-associated gene programs in the dermis of conventionally raised (CR) mice compared with germ-free (GF) counterparts. CR skin exhibited increased collagen content, a thicker dermis, and thicker collagen fibers, indicative of a structurally robust dermal ECM. Similar microbiota-dependent collagen regulation was observed in additional tissues and during embryonic skin development, suggesting contributions from microbiota-derived signals beyond directly colonized sites. Mechanistically, short chain fatty acid butyrate robustly induced epidermal platelet-derived growth factor B (PDGFB) expression in vitro through a histone acetylation-dependent transcriptional program. Conditioned media from butyrate-treated human epidermal equivalents promoted collagen production in primary human dermal fibroblasts, an effect that was attenuated by inhibition of PDGF receptor signaling or histone acetyltransferase activity. Moreover, topical butyrate treatment of explants derived from aged human skin induced epidermal PDGFB and dermal collagen expression. Together, these findings identify a microbiota-epidermis-dermis signaling axis in which the commensal microbiota regulates dermal collagen through metabolite-enhanced epidermal signaling, highlighting a previously underappreciated role for the microbiota in maintaining dermal homeostasis.

Fibroblasts mediate tissue repair after damage, but aberrant fibroblast behavior in response to injury can result in impaired wound healing. Severe burn injury often results in tissue scarring, but the underlying mechanisms by which fibroblasts respond to burn injury and the role of inflammation in fibrosis are not well understood. Here we developed fluorescent reporters of collagen expressing mesenchymal cells enabling real time imaging of fibroblasts during homeostatic development and in response to burn injury using larval zebrafish. We find that fibroblasts derived from the mesenchyme respond to burn injury by engaging in a maturation process, characterized by the expression of vimentin, which is reminiscent of larval development. In burned tissue, fibroblast maturation is perturbed by prolonged neutrophil infiltration, resulting in disorganized extracellular matrix (ECM) and delayed ECM remodeling, which can be rescued by neutrophil depletion. This work adds to our understanding of fibroblast development in zebrafish and shows that collagen expressing mesenchymal cells regulate ECM remodeling in coordination with immune cells during burn wound healing. Summary StatementVimentin-positive fibroblasts are required for normal wound healing in larval zebrafish, but the presence of inflammatory neutrophils after burn injury impairs fibroblast maturation and collagen remodeling.

The innate immune system controls bacterial growth and modulates inflammation during wound healing. TCP-25 is a synthetic thrombin-derived host-defense peptide that combines direct antibacterial activity with neutralization of microbial products and modulation of CD14-dependent inflammatory signaling. We investigated whether this dual mechanism translates to human wounds using longitudinal samples from 24 healthy volunteers enrolled in a randomized, double-blind, within-participant, placebo-controlled phase I dose-escalation study of topical TCP-25 gel in matched epidermal suction blister wounds. We assessed inflammatory cytokines, neutrophil-derived proteins, wound exudation, cultivable bacterial burden, spatial bacterial distribution, and microbiome composition. TCP-25 reduced multiple cytokines, myeloperoxidase, and heparin-binding protein, with the strongest effects observed during the peak inflammatory phase. These changes were accompanied by reduced wound exudation and significant reductions in cultivable bacterial burden. Despite this antibacterial effect, microbiome composition and diversity remained largely unchanged, and participant-specific microbial profiles were preserved. TCP-25 therefore coordinated bacterial control, modulation of the physiological inflammatory response, and reduced wound leakage without major disruption of the resident microbiota composition. These findings provide clinical support for translating nature's endogenous host-defense principles into new therapies for complex wounds.

TGF-{beta} signals through a conserved pathway yet produces diverse outcomes, pointing to regulatory mechanisms upstream of receptor engagement. A family of four latent TGF-{beta} binding proteins (LTBPs) tether pro-TGF-{beta}s and control their localization and activation, but how distinct LTBPs contribute to this regulation is unclear. Here we combine cryo-electron microscopy with functional assays to dissect LTBP-pro-TGF-{beta} interactions. We resolve the LTBP-1/pro-TGF-{beta}1 and LTBP-3/pro-TGF-{beta}3 complex structures, revealing a conserved yet plastic hydrophobic binding interface, and systematically map key residues across all four LTBP and three pro-TGF-{beta} subtypes that determine binding specificity and affinity. Unexpectedly, LTBP-2, previously thought incapable of TGF-{beta} binding, engages pro-TGF-{beta}1 through a covalent linkage via its 16th EGF-like domain, providing a mechanistic link between LTBP-2 function and TGF-{beta} signaling. Beyond tethering, LTBP-3 shifts pro-TGF-{beta}3 from spontaneous activation toward integrin dependence, establishing LTPBs as dynamic modulators that dictate not only where but also how TGF-{beta} is unleashed. Collectively, these findings establish that LTBPs are not redundant ECM scaffolds but a family of functionally distinct regulators that differentially encode TGF-{beta} spatial localization and activation, with implications for isoform-selective therapeutic strategies.

Dorsal skin is widely used in mouse wound-healing, dermatitis, and hair-cycle models, but is often treated as a uniform site. We investigated whether adhesive material (cyanoacrylate)-induced hair regeneration, a model we previously reported, differs by position within the dorsal skin. Adhesive material was applied to four regions along the cranial-to-caudal axis of the mouse dorsal skin. Hair regrowth appeared earlier at the cranial sites than at the caudal sites, and this difference persisted through late anagen and the anagen-to-catagen transition. In contrast, hair regrowth after full-thickness skin excision was slower, smaller in area, and less reproducible than that after adhesive material application. Expression of Hox genes, including Hoxa9, Hoxb9, Hoxc9, Hoxa10, and Hoxc10, was higher at the caudal sites than at the cranial sites but did not correlate with the timing of hair regrowth. RNA sequencing of intact skin from the cranial and caudal sites revealed distinct baseline profiles, including differences in the Wnt inhibitor Sfrp4 and the adipogenic genes Ppar{gamma} and Fabp4. Early after adhesive material application, histological changes and the expression of inflammation- and tissue-repair-related genes also differed between the cranial and caudal skin. These findings indicate that mouse dorsal skin is not a uniform experimental field and that cranial-to-caudal position should be considered when designing and interpreting hair-regeneration and wound-healing experiments in mice.

Matrix metalloproteinase 13 (MMP13) is a zinc-dependent protease that plays key roles in extracellular matrix remodeling. Like several other MMPs, MMP13 has been shown to interact with heparan sulfate (HS), a highly sulfated glycosaminoglycan found at the cell surface and in the extracellular matrix, but the significance of the interaction remains unknown. Here we report that while zymogen and mature forms of MMP13 both bind HS with high affinity, their interactions with HS display markedly different characteristics in terms of preferred HS structure and binding kinetics. By structure-guided mutagenesis, we identified a large HS-binding site of MMP13 consists of 10 residues in the hemopexin domain, 3 residues in the catalytic domain, and 2 residues in the linker region. While these basic residues participate in binding to both zymogen and mature forms of MMP13, the relative contribution of many residues differs substantially between the two forms, which likely contributes to their distinct HS-binding characteristics. Binding of HS to mature MMP13 resulted in selective inhibition of the collagenase activity of MMP13 in a length- and sulfation-dependent manner, but the binding had no effect on degradation of non-collagen substrates. Mechanistically, the inhibitory effect of HS likely results from reduced interdomain flexibility after binding of HS, and/or HS-induced dimerization of MMP13. In sum, our study establishes HS as a multifaceted regulator of MMP13 activity, and discovers that the HS-binding site of MMP13 is a novel exosite that can be targeted to inhibits its collagenase activity.

Fibrosis severity is routinely inferred from collagen abundance, although whether collagen quantity determines pathological fibrosis remains unclear. In Duchenne muscular dystrophy (DMD), chronic muscle injury and inflammation drive extracellular matrix accumulation, making these processes difficult to disentangle. We exploit sarcospan overexpression in mdx mice, a model of DMD (mdxTG), which improves membrane integrity and muscle function despite persistent matrix remodeling. mdxTG muscle accumulates more collagen than mdx yet lacks its dense macrophage-rich scars. Matrisome proteomics and spatial transcriptomics reveal compositionally and spatially distinct matrix states, while decellularized mdxTG matrix protects myotubes from membrane damage relative to mdx matrix. Despite these differences, both dystrophic matrices remain stiff and induce nuclear YAP in fibro-adipogenic progenitors. Verteporfin suppresses collagen production and reduces fibrosis in vivo, while nuclear YAP is increased in FAPs from patients with DMD. Thus, collagen abundance alone does not define pathological fibrosis; matrix organization, biological activity, and mechanosignaling distinguish functionally distinct fibrotic states.

Tendon injuries often result in fibrosis, compromising function and predisposing to re-injury. Here, we used a full-width, non-repair Achilles tendon transection model in young (3 weeks old) and adult (18-20 weeks) rats to elucidate the cellular mechanisms governing regenerative versus fibrotic healing. Functional and biomechanical analyses revealed that young tendons recovered motion and load-bearing capacity more rapidly but exhibited more fibrotic early healing. Single-nuclei RNA sequencing identified seven major cell populations within the connective tissue compartment. Adult tendons maintained a "synthetic fibroblast" population marked by upregulated ECM synthesis, reduced stress-related gene expression, whereas young tendons favored expansion of Cxcl12/Lrp6/Gas6 fibrotic fibroblasts linked to oxidative and pro-angiogenic signaling. The young group showed sustained activation of Nox4-Gas6 pathways driving a self-reinforcing fibrotic circuit. These findings define a fibroblast lineage bifurcation that dictates oxidative stress signaling as a key regulator of fibrotic remodeling, highlighting potential therapeutic targets to promote regenerative tendon repair.

Fibroblast transcriptomic states reflect physiological and pathological tissue contexts, yet the upstream determinants that stabilize these states remain poorly defined. Integrins mediate extracellular matrix (ECM) adhesion and biochemical signaling, but whether they encode mechanical constraints into stable transcriptomic programs is unclear. Using engineered mouse fibroblasts, bulk and single-cell transcriptomics, and controlled micromechanical confinement, we show that integrins can shape the transcriptomic landscape. The bulk transcriptome of fibroblasts expressing V- and {beta}1-class indicates a shared mechanosensitive baseline, except when these integrin classes are expressed individually. We also found integrin-specific gene clusters, including {beta}1-class integrin-dependent enrichment of Areg, Epha7, Lhpp and Igf2r, which regulate development, regeneration and disease, and altered YAP1 targeted gene expression. At single-cell resolution under confinement, {beta}1-class integrins sustain a progenitor-associated program, whereas their loss or V-class enrichment promotes a constitutively activated state linked to injury repair and wound healing. Mechanical confinement and confinement duration further reshapes these states in an integrin-identity-dependent manner. Our findings establish integrin-identity as a determinant of how fibroblasts transduce mechanotemporal inputs from the cell surface to the nucleus.

The complex interplay between extracellular matrix stiffness and actomyosin contractility regulates long-term integrin-based adhesion and signaling in mammalian cells. However, how cells sense and respond to the stiffness of the environment during the first minutes of initiating adhesion remains elusive. Here, we show that fibroblasts upon initiating adhesion to fibronectin switch between two distinct mechanosensitive adhesion modes. The first mode of "slow" adhesion strengthening, shared between 5{beta}1 and v{beta}3 integrins and depends modestly on fibronectin stiffness. Fibroblasts adapt this slow adhesion strengthening mode, which is independent of actomyosin contractility and intracellular signaling, on soft fibronectin substrates (<5 kPa). On stiff fibronectin substrates (>5 kPa), however, 5{beta}1 integrins but not v{beta}3 integrins switch to a "fast" adhesion strengthening mode that considerably strengthens adhesion within seconds. The switch to the fast mode depends on myosin II-mediated contractility and a mechanosensitive signaling hub that includes the 5{beta}1 integrin-FN catch bond, paxillin, and focal adhesion kinase. The mechanistic findings highlight the similarities and differences of integrin-type specific adhesion strengthening and intracellular regulation, which depend on mechanotransduction in fibroblasts during adhesion initiation.

Extracellular matrix (ECM) remodeling and angiogenesis are essential processes underlying endometrial decidualization during embryo implantation. Fibrillar collagens, the major structural components of the ECM, form the architectural framework of the decidua and undergo dynamic reorganization during this process. However, the functional role of type V collagen--a key regulator of collagen fibrillogenesis-- remains undefined. Here, we demonstrate that Col5a1 is highly expressed in decidual stromal and endothelial cells of the mouse uterus. Conditional deletion of Col5a1 leads to progressive uterine hemorrhage beginning at gestation day 8, culminating in complete embryo resorption and pregnancy loss by day 12. Col5a1-deficient decidua exhibits severe structural distortion, marked disorganization of fibrillar collagen, shallow and misdirected embryo invasion, and profound disruption of decidual angiogenesis and vascular network formation. Transcriptomic profiling further reveals distinct gene expression signatures and signaling pathways regulated by COL5A1 in the decidua. Collectively, these findings identify type V collagen as a critical ECM regulator required for maintaining decidual integrity, supporting angiogenesis, and ensuring successful pregnancy.

Regulated keratinocyte differentiation is required for formation of the stratified epidermis and a functional barrier. Understanding genetic drivers of keratinocyte differentiation is crucial for understanding several skin diseases. Perturb-seq is a single-cell CRISPR screen that measures transcriptomic responses to perturbations. To date, Perturb-seq experiments have principally focused on 2-dimensional cell culture models lacking hallmarks of skin development - physiological desmosome formation and barrier function. Here, we leverage Perturb-seq in an epidermal organoid model that recapitulates physiologically relevant differentiation programs. We demonstrate that our perturbations significantly impact diverse differentiation programs and reveal bidirectional function of non-canonical NF-{kappa}B signaling in late keratinocyte differentiation.

Although microtubules (MT) are established regulators of striated muscle mechanics, how MT lattice organization and post-translational modifications (PTM) individually shape contractility in healthy skeletal muscle fibers remains incompletely understood. We used an ex vivo single muscle fiber culture under unloading, examining acetylation and detyrosination (deTyr). During long-term 2D culture, the MT lattice was disrupted by transverse MT depletion without changes in MT abundance. In individual fibers, MT structure, but not abundance, positively correlated with sarcomere shortening non-linearly. When fibers were cultured in 3D hydrogels, the MT lattice was similarly disrupted yet shortening was preserved, with increased longitudinal MTs and decreased deTyr-MTs. Pharmacologically, contractility increased with either a decrease (parthenolide) or an increase (Taxol) in deTyr-MTs, and Taxol further rescued the MT lattice. Our findings identify MT organization and detyrosination, rather than MT abundance, as key determinants of muscle fiber contractility, positioning deTyr-MT as a load-responsive, bidirectional marker relevant to disuse atrophy and aging.

Synovial joints like the knee are home to adipose tissue depots whose anatomy and functions are closely intertwined with that of other intra-articular soft tissues such as synovium, underscoring the growing understanding that joints are multi-tissue organs. Traumatic joint injury and the onset of osteoarthritis (OA) dramatically remodel the intra-articular adipose niche, marked by infiltration of fibrotic tissue postulated to underpin OA-associated joint stiffness and pain, yet we know very little about the disease-associated dynamics of joint adipose remodeling nor the mechanisms driving these phenomena. Here, we employed 2D histomorphometry and spatial transcriptomics, alongside 3D osmium tetroxide-enhanced micro-computed tomography to comprehensively define the spatiotemporal, structural, and transcriptional rewiring of joint adipose tissue in a non-invasive mouse model of post-traumatic osteoarthritis (PTOA). These revealed marked loss of intra-articular adiposity accompanied by expansion of fibroblast-rich, collagen-dense tissue with pro-fibrotic hallmarks and Wnt/{beta}-catenin-enriched gene programs. Joint adipose exhibited a distinct transcriptional signature compared to subcutaneous white adipose tissue, pointing to unique, depot-specific functions. Stromal cells isolated from PTOA joints had heightened baseline expression of fibrotic and Wnt pathway genes and exhibited impaired de novo adipogenesis, in contrast to cells derived from healthy joints. In accordance with the destabilized biomechanics of PTOA joints, in vitro modeling demonstrated that prolonged, injurious loading and perturbed Wnt/{beta}-catenin signaling were convergent anti-adipogenic cues that suppressed lipid droplet formation and adipogenic gene induction, while promoting markers of fibrosis in joint-derived stromal cells. Complementary gain-of-function studies using ex vivo joint adipose explants and in vivo joint injections demonstrated that chronic Wnt/{beta}-catenin activation, as seen in OA joints, is sufficient to diminish the intra-articular adipogenic program and shift adipose to a more fibrotic phenotype, independent of joint injury. Collectively, these findings establish a multi-modal framework for quantifying joint adipose atrophy and implicate aberrant Wnt/{beta}-catenin signaling and pathological mechanical loading as key factors impairing de novo adipogenesis and driving fibrotic remodeling of intra-articular adipose tissue in PTOA.

Structural AbstractO_ST_ABSBackgroundC_ST_ABSTriple-negative breast cancer (TNBC) represents a major clinical challenge due to its aggressiveness, heterogeneity and limited availability of effective targeted therapy. We investigated whether LRP-1, a multifunctional cell-surface endocytic and signaling receptor, contributes to TNBC progression. MethodsUsing CRISPR-Cas9, LRP-1-deficient murine 4T1 and human HS578-T TNBC cells were used. Functional consequences were assessed through migration, invasion, and 3D spheroid assays, imaging of focal adhesions and actin organization, atomic force microscopy, and plasmin activity assays. Global molecular reprogramming was analyzed by label-free quantitative proteomics and secretomics. LRP-1-deficient or proficient 4T1 cells were implanted orthotopically in immunocompetent mice; tumor progression was monitored longitudinally while peritumoral collagen architecture and immune microenvironment composition were characterized by second harmonic generation imaging and immunohistochemistry. ResultsWe show that LRP-1 loss reduces TNBC aggressiveness, as reflected by decreased migration and invasive capacity, reduced spheroid evasion, and significant morphological changes in focal adhesion and actin structure. LRP-1-deficient cells became stiffer and showed lower LOXL-4 levels, while pericellular proteolytic activity remained unchanged, suggesting other proteases mechanism. Multi-omic analysis revealed alterations in extracellular matrix (ECM), epithelial-mesenchymal transition, and inflammatory pathways. In vivo, LRP-1-deficiency reduced tumor progression and peritumoral collagen deposition, while increasing CD8+ T and Natural Killer cell infiltration, together with a cytokine profiling compatible with a more immune-permissive microenvironment. ConclusionsLRP-1 act as a key contributor in TNBC progression through matrix remodeling, mechano-adaptation, and immune exclusion. Positioning it as a candidate biomarker for TNBC patients who are likely to benefit from stroma-targeting therapies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/732906v2_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@1b595c2org.highwire.dtl.DTLVardef@7b208aorg.highwire.dtl.DTLVardef@1956e54org.highwire.dtl.DTLVardef@17e55d0_HPS_FORMAT_FIGEXP M_FIG C_FIG

An autocrine pro-adhesive/pro-contractile signaling loop, through the mechanosensitive transcriptional cofactor YAP, promotes fibrosis. The CCN family of matricellular proteins modify adhesive signaling. Of these, CCN3 is antifibrotic. We show that BLR-200, a CCN3-derived peptide, has anti-fibrotic properties in the bleomycin-induced model of scleroderma skin fibrosis. In vitro, BLR-200 delayed, but did not abolish, fibroblast adhesion to collagen and nuclear YAP localization. In vivo, BLR-200 prevented/treated bleomycin-induced skin fibrosis, and reduced bleomycin-induced expression of profibrotic genes including alpha-smooth muscle actin, CCN1 and CCN2. Lineage tracing and scRNA-seq analyses revealed that the myofibroblasts in this model were quantitatively derived from collagen-lineage Pi16+/Col15+ve fibroblasts. BLR-200 prevented myofibroblast differentiation in this model and trajectory of fibroblasts toward a Sfrp2-positive subset, a cell type associated with poor clinical outcome. BLR-200 impairs YAP activation in vitro and appearance of translationally-relevant fibroblast subtypes in vivo and is a novel anti-fibrotic agent for SSc skin fibrosis.

Osteoblastic bone metastasis, in which disseminated tumor cells drive net bone formation, is a clinically distinct and mechanistically underexplored form of skeletal disease that is enriched in hormone receptor-positive breast cancers. Preclinical models of bone metastasis from breast cancer predominantly rely on immunodeficient hosts inoculated with osteolytic human breast cancer cell lines, limiting the study of immune-dependent mechanisms of bone remodeling. Here we describe the development and characterization of an immunocompetent, syngeneic osteoblastic bone metastasis model using intratibial injection of PyMT-CK(OB), a luciferase-expressing derivative of the MMTV-PyMT mammary carcinoma cell line, in FVB/N mice. PyMT-CK(OB) cells produced detectable bioluminescent signal after intratibial injection, enabling longitudinal monitoring of tumor progression. Micro-computed tomography (microCT) revealed significant increases in trabecular bone volume fraction and trabecular number at three and four weeks post-injection, consistent with osteoblastic remodeling. Histological analysis confirmed dense bone lesion formation in tumor-bearing bones. Critically, this osteoblastic phenotype was entirely absent in immunodeficient NOD SCID hosts, despite robust tumor growth, supporting a role for immune competence in tumor-induced bone formation. Loss of bioluminescent signal in immunocompetent mice reflected either immune pressure on reporter gene expression or limited space for cancer cell expansion in the bone, rather than tumor regression or hypoxia, as confirmed by hypoxia imaging and histological endpoint analysis. In contrast, a second PyMT cell subline, PyMT-CF, maintained sustained bioluminescent signal and produced predominantly osteolytic lesions, providing a complementary syngeneic model of osteolytic disease from the same parental background. In vitro hydrogel coculture experiments and protein array analysis of conditioned media revealed that the PyMT sublines have differing impact on MC3T3 osteoblast mineralization, identifying candidate mediators of divergent bone remodeling phenotypes. R7 mammary carcinoma cells derived from MMTV-RON transgenic mouse mammary tumors did not induce measurable bone remodeling under equivalent experimental conditions. Together, these models provide a validated, immunologically intact framework for studying the mechanistic basis of osteoblastic bone metastasis and evaluating therapeutic interventions targeting the tumor-bone microenvironment.