Cognate Receptor Research Articles

CRD: A de novo design algorithm for the prediction of cognate protein receptors for small molecule ligands

While predicting a ligand that binds to a protein is feasible with current methods, the opposite, i.e., the prediction of a receptor for a ligand remains challenging. We present an approach for predicting receptors of a given ligand that uses de novo design and structural bioinformatics. We have developed the algorithm CRD, comprising multiple modules combining fragment-based sub-site finding, a machine learning function to estimate the size of the site, a genetic algorithm that encodes knowledge on protein structures and a physics-based fitness scoring scheme. CRD includes a pseudo-receptor design component followed by a mapping component to identify proteins that might contain these sites. CRD recovers the sites and receptors of several natural ligands. It designs similar sites for similar ligands, yet to some extent can distinguish between closely related ligands. CRD correctly predicts receptor classes for several drugs and might become a valuable tool for drug discovery.

Dimerization of the 4Ig isoform of B7-H3 in tumor cells mediates enhanced proliferation and tumorigenic signaling

B7-H3 (CD276) has two isoforms (2Ig and 4Ig), no confirmed cognate receptor, and physiological functions that remain elusive. While differentially expressed on many solid tumors correlating with poor survival, mechanisms of how B7-H3 signals in cis (tumor cell) versus in trans (immune cell co-regulator) to elicit pro-tumorigenic phenotypes remain poorly defined. Herein, we characterized a tumorigenic and signaling role for tumor cell-expressed 4Ig-B7-H3, the dominant human isoform, in gynecological cancers that could be abrogated upon CRISPR/Cas9 knockout of B7-H3; tumorigenesis was rescued upon re-expression of 4Ig-B7-H3. Size exclusion chromatography revealed dimerization states for the extracellular domains of both human 4Ig- and murine 2Ig-B7-H3. mEGFP lifetimes of expressed 4Ig-B7-H3-mEGFP fusions determined by FRET-FLIM assays confirmed close-proximity interactions of 4Ig-B7-H3 and identified two distinct homo-FRET lifetime populations, consistent with monomeric and homo-dimer interactions. In live cells, bioluminescence imaging of 4Ig-B7-H3-mediated split luciferase complementation showed dimerization of 4Ig-B7-H3. To separate basal from dimer state activities in the absence of a known receptor, C-terminus (cytosolic) chemically-induced dimerization of 4Ig-B7-H3 increased tumor cell proliferation and cell activation signaling pathways (AKT, Jak/STAT, HIF1α, NF-κβ) significantly above basal expression of 4Ig-B7-H3 alone. These results revealed a new, dimerization-dependent intrinsic tumorigenic signaling role for 4Ig-B7-H3, likely acting in cis, and provide a therapeutically-actionable target for intervention of B7-H3-dependent tumorigenesis.

CCL16 is a pro-tumor chemokine that recruits monocytes and macrophages to promote hepatocellular carcinoma progression.

Intricate signaling cascades involving chemokines and their cognate receptors on neoplastic and immune constituents within tumor microenvironment have garnered substantial research interest. Our investigation delineates the contribution of Chemokine (C-C motif) ligand 16 (CCL16) to the clinico-pathological features and tumorigenesis of hepatocellular carcinoma (HCC). Analysis of 237 pairs of HCC specimens unraveled a significant association between CCL16 expression and vascular invasion, early-stage clinicopathological features, and diminished recurrence-free survival among HCC patients. Immunohistochemical (IHC) assays of the clinical HCC specimens indicated elevated CCL16 in tumorous versus normal hepatic tissues. Our in vivo experiments demonstrated CCL16 overexpression fostered tumor proliferation, whereas in vitro assays elucidated that CCL16-mediated chemotactic recruitment of monocytes and M2 macrophages was orchestrated via CCR1 and CCR5. In contrast to previous claims that CCL16 is physiologically irrelevant and has minimal affinity for its receptors (CCR1, CCR2, CCR5, CCR8), our findings unravel that inhibition of CCL16/CCR1 and CCL16/CCR5 interactions through receptor-specific antagonists markedly impeded CCL16-directed chemotaxis, migration, adhesion, and leukocyte recruitment. Moreover, CCL16-overexpression in HCCs significantly augmented levels of several cytokines implicated in tumor progression, namely IL-6, IL-10 and VEGFA. IHC analysis of CCL16-overexpressing xenografts elicited greatly enhanced levels of VEGFA and IL-6, while assessments of HCC specimens confirmed a positive correlation between CCL16 expression and IL-6 and VEGFA levels. Collectively, our study highlights oncogenic role of CCL16 in hepatocarcinogenesis and provides a foundational basis for novel therapeutic interventions targeting the CCL16/CCR1/CCR5 axis.

Enpp2 haploinsufficiency induces an eye-open-at-birth phenotype in the DBA/2 background.

Autotaxin, encoded by the Enpp2 gene, produces lysophosphatidic acid (LPA), which exerts numerous biological functions via its cognate receptors. Enpp2 null mutant mice die by embryonic day 9.5 owing to aberrant vascular development in the yolk sac, preventing analysis after that period. In this study, we found that Enpp2 heterozygous mice in the DBA/2 genetic background showed an eye-open-at-birth phenotype at high frequency, caused by failure of eyelid closure during the embryonic stage. Notably, wildtype pups from the Enpp2 heterozygous dam showed the phenotype, although at lower frequency, suggesting that maternal LPA affects the embryonic development.

Non-enzymatic glycation and diabetic kidney disease.

Chronic diabetes leads to various complications including diabetic kidney disease (DKD). DKD is a major microvascular complication and the leading cause of morbidity and mortality in diabetic patients. Varying degrees of proteinuria and reduced glomerular filtration rate are the cardinal clinical manifestations of DKD that eventually progress into end-stage renal disease. Histopathologically, DKD is characterized by renal hypertrophy, mesangial expansion, podocyte injury, glomerulosclerosis, and tubulointerstitial fibrosis, ultimately leading to renal replacement therapy. Amongst the many mechanisms, hyperglycemia contributes to the pathogenesis of DKD via a mechanism known as non-enzymatic glycation (NEG). NEG is the irreversible conjugation of reducing sugars onto a free amino group of proteins by a series of events, resulting in the formation of initial Schiff's base and an Amadori product and to a variety of advanced glycation end products (AGEs). AGEs interact with cognate receptors and evoke aberrant signaling cascades that execute adverse events such as oxidative stress, inflammation, phenotypic switch, complement activation, and cell death in different kidney cells. Elevated levels of AGEs and their receptors were associated with clinical and morphological manifestations of DKD. In this chapter, we discussed the mechanism of AGEs accumulation, AGEs-induced cellular and molecular events in the kidney and their impact on the pathogenesis of DKD. We have also reflected upon the possible options to curtail the AGEs accumulation and approaches to prevent AGEs mediated adverse renal outcomes.

Structural and functional characterization of peste des petits ruminants virus coded hemagglutinin protein using various in-silico approaches.

Peste des petits ruminants (PPR), a disease of socioeconomic importance has been a serious threat to small ruminants. The causative agent of this disease is PPR virus (PPRV) which belongs to the genus Morbillivirus. Hemagglutinin (H) is a PPRV coded transmembrane protein embedded in the viral envelope and plays a vital role in mediating the entry of virion particle into the cell. The infected host mounts an effective humoral response against H protein which is important for host to overcome the infection. In the present study, we have investigated structural, physiological and functional properties of hemagglutinin protein using various computational tools. The sequence analysis and structure prediction analysis show that hemagglutinin protein comprises of beta sheets as the predominant secondary structure, and may lack neuraminidase activity. PPRV-H consists of several important domains and motifs that form an essential scaffold which impart various critical roles to the protein. Comparative modeling predicted the protein to exist as a homo-tetramer that binds to its cognate cellular receptors. Certain amino acid substitutions identified by multiple sequence alignment were found to alter the predicted structure of the protein. PPRV-H through its predicted interaction with TLR-2 molecule may drive the expression of CD150 which could further propagate the virus into the host. Together, our study provides new insights into PPRV-H protein structure and its predicted functions.

The role of pituitary adenylate cyclase-activating polypeptide neurons in the hypothalamic ventromedial nucleus and the cognate PAC1 receptor in the regulation of hedonic feeding.

Obesity is a health malady that affects mental, physical, and social health. Pathology includes chronic imbalance between energy intake and expenditure, likely facilitated by dysregulation of the mesolimbic dopamine (DA) pathway. We explored the role of pituitary adenylate cyclase-activating polypeptide (PACAP) neurons in the hypothalamic ventromedial nucleus (VMN) and the PACAP-selective (PAC1) receptor in regulating hedonic feeding. We hypothesized that VMN PACAP neurons would inhibit reward-encoding mesolimbic (A10) dopamine neurons via PAC1 receptor activation and thereby suppress impulsive consumption brought on by intermittent exposure to highly palatable food. Visualized whole-cell patch clamp recordings coupled with in vivo behavioral experiments were utilized in wildtype, PACAP-cre, TH-cre, and TH-cre/PAC1 receptor-floxed mice. We found that bath application of PACAP directly inhibited preidentified A10 dopamine neurons in the ventral tegmental area (VTA) from TH-cre mice. This inhibitory action was abrogated by the selective knockdown of the PAC1 receptor in A10 dopamine neurons. PACAP delivered directly into the VTA decreases binge feeding accompanied by reduced meal size and duration in TH-cre mice. These effects are negated by PAC1 receptor knockdown in A10 dopamine neurons. Additionally, apoptotic ablation of VMN PACAP neurons increased binge consumption in both lean and obese, male and female PACAP-cre mice relative to wildtype controls. These findings demonstrate that VMN PACAP neurons blunt impulsive, binge feeding behavior by activating PAC1 receptors to inhibit A10 dopamine neurons. As such, they impart impactful insight into potential treatment strategies for conditions such as obesity and food addiction.

Detection of Beta-Glucan Contamination in Nanoparticle Formulations.

Beta-glucans with diverse chemical structures are produced by a variety of microorganisms and are commonly found in microbial cell walls. β-(1,3)-D-glucans are present in yeast and fungi, and, for this reason, their traces are commonly used as a sign of yeast or fungal infection or contamination. Despite being less immunologically active than endotoxins, beta-glucans are pro-inflammatory and can activate cytokines and other immunological responses via their cognate pattern recognition receptors. Unlike endotoxins, there is no established threshold pyrogen dose for beta-glucans; as such, their quantity in pharmaceutical products is not regulated. Nevertheless, regulatory agencies recognize the potential contribution of beta-glucans to the immunogenicity of protein-containing drug products and recommend assessing beta-glucans to aid the interpretation of immunotoxicity studies and assess the risk of immunogenicity. The protocol for the detection and quantification of β-(1,3)-D-glucans in nanoparticle formulations is based on a modified limulus amoebocyte lysate assay. The results of this test are used to inform immunotoxicity studies of nanotechnology-based drug products.

Nanoparticles in prevention of protein glycation.

Advanced glycation end products (AGEs) are formed by the non-enzymatic attachment of carbohydrates to a biological macromolecule. These AGEs bind to their cognate receptor called receptor forAGEs (RAGEs), which becomes one of the important causal factors for the initiation and progression of several diseases. A deep understanding into the pathways of RAGEs will help in identifying novel intervention modalities as a part of new therapeutic strategies. Although several approaches exist to target this pathway using small molecules, compounds of plant origin etc, nanoparticles have proven to be a critical method, given its several advantages. A high bioavailability, biocompatibility, ability to cross blood brain barrier and modifiable surface properties give nanoparticles an upper edge over other strategies. In this chapter, we will discuss AGEs, their involvement in diseases and the nanoparticles used for targeting this pathway.

The chemokine receptor CCR8 is not a high-affinity receptor for the human chemokine CCL18.

The primate-specific chemokine CCL18 is a potent chemoattractant for T cells and is expressed at elevated levels in several inflammatory diseases. However, the cognate receptor for CCL18 remains unconfirmed. Here, we describe attempts to validate a previous report that the chemokine receptor CCR8 is the human CCL18 receptor (Islam et al. J Exp Med. 2013, 210:1889-98). Two mouse pre-B cell lines (4DE4 and L1.2) exogenously expressing CCR8 exhibited robust migration in response to the known CCR8 ligand CCL1 but not to CCL18. Similarly, CCL1 but not CCL18 induced internalization of CCR8 on 4DE4 cells. CCR8 expressed on Chinese hamster ovarian (CHO) cells mediated robust G protein activation, inhibition of cAMP synthesis and β-arrestin2 recruitment in response to CCL1 but not CCL18. Several N- and C-terminal variants of CCL18 also failed to stimulate CCR8 activation. On the other hand, and as previously reported, CCL18 inhibited CCL11-stimulated migration of 4DE4 cells expressing the receptor CCR3. These data suggest that CCR8, at least in the absence of unidentified cofactors, does not function as a high affinity receptor for CCL18.

The BET inhibitor apabetalone decreases neuroendothelial proinflammatory activation in vitro and in a mouse model of systemic inflammation.

Brain vascular inflammation is characterized by endothelial activation and immune cell recruitment to the blood vessel wall, potentially causing a breach in the blood - brain barrier, brain parenchyma inflammation, and a decline of cognitive function. The clinical-stage small molecule, apabetalone, reduces circulating vascular endothelial inflammation markers and improves cognitive scores in elderly patients by targeting epigenetic regulators of gene transcription, bromodomain and extraterminal proteins. However, the effect of apabetalone on cytokine-activated brain vascular endothelial cells (BMVECs) is unknown. Here, we show that apabetalone treatment of BMVECs reduces hallmarks of in vitro endothelial activation, including monocyte chemoattractant protein-1 (MCP-1) and RANTES chemokine secretion, cell surface expression of endothelial cell adhesion molecule VCAM-1, as well as endothelial capture of THP-1 monocytes in static and shear stress conditions. Apabetalone pretreatment of THP-1 downregulates cell surface expression of chemokine receptors CCR1, CCR2, and CCR5, and of the VCAM-1 cognate receptor, integrin α4. Consequently, apabetalone reduces THP-1 chemoattraction towards soluble CCR ligands MCP-1 and RANTES, and THP-1 adhesion to activated BMVECs. In a mouse model of brain inflammation, apabetalone counters lipopolysaccharide-induced transcription of endothelial and myeloid cell markers, consistent with decreased neuroendothelial inflammation. In conclusion, apabetalone decreases proinflammatory activation of brain endothelial cells and monocytes in vitro and in the mouse brain during systemic inflammation.

Isthmin-1: A critical regulator of branching morphogenesis and metanephric mesenchyme condensation during early kidney development.

Isthmin-1 (Ism1) was first described to be syn-expressed with Fgf8 inXenopus. However, its biological role has not been elucidated until recent years. Despite of accumulated evidence that Ism1 participates in angiogenesis, tumor invasion, macrophage apoptosis, and glucose metabolism, the cognate receptors for Ism1 remain largely unknown. Ism1 deficiency in mice results in renal agenesis (RA) with a transient loss of Gdnf transcription and impaired mesenchyme condensation at E11.5. Ism1 binds to and activates Integrin α8β1 to positively regulate Gdnf/Ret signaling, thus promoting mesenchyme condensation and ureteric epithelium branching morphogenesis. Here, we propose the hypothesis underlying the mechanism by which Ism1 regulates branching morphogenesis during early kidney development.

Extracellular pectin-RALF phase separation mediates FERONIA global signaling function

The FERONIA (FER)-LLG1 co-receptor and its peptide ligand RALF regulate myriad processes for plant growth and survival. Focusing on signal-induced cell surface responses, we discovered that intrinsically disordered RALF triggers clustering and endocytosis of its cognate receptors and FER- and LLG1-dependent endocytosis of non-cognate regulators of diverse processes, thus capable of broadly impacting downstream responses. RALF, however, remains extracellular. We demonstrate that RALF binds the cell wall polysaccharide pectin. They phase separate and recruit FER and LLG1 into pectin-RALF-FER-LLG1 condensates to initiate RALF-triggered cell surface responses. We show further that two frequently encountered environmental challenges, elevated salt and temperature, trigger RALF-pectin phase separation, promiscuous receptor clustering and massive endocytosis, and that this process is crucial for recovery from stress-induced growth attenuation. Our results support that RALF-pectin phase separation mediates an exoskeletal mechanism to broadly activate FER-LLG1-dependent cell surface responses to mediate the global role of FER in plant growth and survival.

PGF2α signaling drives fibrotic remodeling and fibroblast population dynamics in mice.

Idiopathic pulmonary fibrosis (IPF) is a chronic parenchymal lung disease characterized by repetitive alveolar cell injury, myofibroblast proliferation, and excessive extracellular matrix deposition for which unmet need persists for effective therapeutics. The bioactive eicosanoid, prostaglandin F2α, and its cognate receptor FPr (Ptgfr) are implicated as a TGF-β1-independent signaling hub for IPF. To assess this, we leveraged our published murine PF model (IER-SftpcI73T) expressing a disease-associated missense mutation in the surfactant protein C (Sftpc) gene. Tamoxifen-treated IER-SftpcI73T mice developed an early multiphasic alveolitis and transition to spontaneous fibrotic remodeling by 28 days. IER-SftpcI73T mice crossed to a Ptgfr-null (FPr-/-) line showed attenuated weight loss and gene dosage-dependent rescue of mortality compared with FPr+/+ cohorts. IER-SftpcI73T/FPr-/- mice also showed reductions in multiple fibrotic endpoints for which administration of nintedanib was not additive. Single-cell RNA-Seq, pseudotime analysis, and in vitro assays demonstrated Ptgfr expression predominantly within adventitial fibroblasts, which were reprogrammed to an "inflammatory/transitional" cell state in a PGF2α /FPr-dependent manner. Collectively, the findings provide evidence for a role for PGF2α signaling in IPF, mechanistically identify a susceptible fibroblast subpopulation, and establish a benchmark effect size for disruption of this pathway in mitigating fibrotic lung remodeling.

The β-Blocker Carvedilol and Related Aryloxypropanolamines Promote ERK1/2 Phosphorylation in HEK293 Cells with K A Values Distinct From Their Equilibrium Dissociation Constants as β 2-Adrenoceptor Antagonists: Evidence for Functional Affinity.

The determination of affinity by using functional assays is important in drug discovery because it provides a more relevant estimate of the strength of interaction of a ligand to its cognate receptor than radioligand binding. However, empirical evidence for so-called, "functional affinity" is limited. Herein, we determined whether the affinity of carvedilol, a β-adrenoceptor antagonist used to treat heart failure that also promotes extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation, differed between these two pharmacological activities. Four structurally related β-adrenoceptor antagonists (alprenolol, carazolol, pindolol, propranolol) that also activated ERK1/2 were included as comparators to enhance our understanding of how these drugs work in the clinical setting. In HEK293 cells stably expressing the human β 2-adrenoceptor carvedilol and related aryloxypropanolamines were partial agonists of ERK1/2 phosphorylation with potencies ([A]50s) that were lower than their equilibrium dissociation constants (K Bs) as β 2-adrenoceptor antagonists. As the [A]50 of a partial agonist is a good approximation of its K B, then these data indicated that the affinities of carvedilol and related ligands for these two activities were distinct. Moreover, there was a significant negative rank order correlation between the [A]50 of each ligand to activate ERK1/2 and their intrinsic activities (i.e., as intrinsic activity for ERK1/2 phosphorylation increased, so did affinity). Genome editing revealed that the transducer that coupled the β 2-adrenoceptor to ERK1/2 phosphorylation in response to carvedilol and other β 2-adrenoceptor antagonists was Gαs. Collectively, these data support the concept of "functional affinity" and indicate that the ability of the β 2-adrenoceptor to recruit Gαs may influence the affinity of the activating ligand. SIGNIFICANCE STATEMENT: In HEK293 cells overexpressing the human β2-adrenoceptor carvedilol and four related aryloxypropanolamines behaved as β2-adrenoceptor antagonists and partial agonists of ERK1/2 phosphorylation with rank orders of affinity that were distinct. These data imply that carvedilol and other β-blockers can stabilize the β2-adrenoceptor in different affinity conformations that are revealed when functionally distinct responses are measured. This is the basis for the pharmacological concept of "functional affinity."

Gas6-Axl Signaling Induces SRF/MRTF-A Gene Transcription via MICAL2.

MICAL2 is an actin-regulatory protein that functions through redox modification of actin. Nuclear localized MICAL2 triggers the disassembly of nuclear actin, which subsequently leads to nuclear retention of the actin-binding transcriptional coregulator myocardin-related transcription factor-A (MRTF-A), which leads to the activation of serum response factor (SRF)/MRTF-A-dependent gene transcription. In this study, we show that the secreted signaling protein GAS6 (growth-arrest specific 6) and its cognate receptor Axl, a transmembrane tyrosine kinase, also induce the activation of SRF/MRTF-A and their downstream target genes. We find that serum-induced SRF/MRTF-A-dependent gene expression can be blocked, in part, by the inhibition of Axl signaling. Furthermore, we find that Gas6/Axl-induced SRF/MRTF-A-dependent transcription is dependent on MICAL2. Gas6/Axl promotes cell invasion, which is blocked by MICAL2 knockdown, suggesting that MICAL2 promotes cytoskeletal effects of the Gas6/Axl pathway. We find that Gas/6/Axl signaling promotes the nuclear localization of MICAL2, which may contribute to the ability of Gas6/SRF to augment SRF/MRTF-A-dependent gene transcription. The physiological significance of the Gas6/Axl-MICAL2 signaling pathway described here is supported by the marked gene expression correlation across a broad array of different cancers between MICAL2 and Axl and Gas6, as well as the coexpression of these genes and the known SRF/MRTF-A target transcripts. Overall, these data reveal a new link between Gas6/Axl and SRF/MRTF-A-dependent gene transcription and link MICAL2 as a novel effector of the Gas6/Axl signaling pathway.

CD200/CD200R: Bidirectional Role in Cancer Progression and Immunotherapy.

As an immune checkpoint molecule, CD200 serves a foundational role in regulating immune homeostasis and promoting self-tolerance. While CD200 expression occurs in various immune cell subsets and normal tissues, its aberrant expression patterns in hematologic malignancies and solid tumors have been linked to immune evasion and cancer progression under pathological conditions, particularly through interactions with its cognate receptor, CD200R. Through this CD200/CD200R signaling pathway, CD200 exerts its immunosuppressive effects by inhibiting natural killer (NK) cell activation, cytotoxic T cell functions, and M1-polarized macrophage activity, while also facilitating expansion of myeloid-derived suppressor cells (MDSCs) and Tregs. Moreover, CD200/CD200R expression has been linked to epithelial-to-mesenchymal transition and distant metastasis, further illustrating its role in cancer progression. Conversely, CD200 has also been shown to exert anti-tumor effects in certain cancer types, such as breast carcinoma and melanoma, indicating that CD200 may exert bidirectional effects on cancer progression depending on the specific tumor microenvironment (TME). Regardless, modulating the CD200/CD200R axis has garnered clinical interest as a potential immunotherapeutic strategy for cancer therapy, as demonstrated by early-phase clinical trials. However, further research is necessary to fully understand the complex interactions of CD200 in the tumor microenvironment and to optimize its therapeutic potential in cancer immunotherapy.

The neuroendocrine system of Ciona intestinalis Type A, a deuterostome invertebrate and the closest relative of vertebrates

Deuterostome invertebrates, including echinoderms, hemichordates, cephalochordates, and urochordates, exhibit common and species-specific morphological, developmental, physiological, and behavioral characteristics that are regulated by neuroendocrine and nervous systems. Over the past 15 years, omics, genetic, and/or physiological studies on deuterostome invertebrates have identified low-molecular-weight transmitters, neuropeptides and their cognate receptors, and have clarified their various biological functions. In particular, there has been increasing interest on the neuroendocrine and nervous systems of Ciona intestinalis Type A, which belongs to the subphylum Urochordata and occupies the critical phylogenetic position as the closest relative of vertebrates. During the developmental stage, gamma-aminobutylic acid, D-serine, and gonadotropin-releasing hormones regulate metamorphosis of Ciona. In adults, the neuropeptidergic mechanisms underlying ovarian follicle growth, oocyte maturation, and ovulation have been elucidated. This review article provides the most recent and fundamental knowledge of the neuroendocrine and nervous systems of Ciona, and their evolutionary aspects.

A PTHrP Gradient Drives Mandibular Condylar Chondrogenesis via Runx2

The mandibular condylar cartilage (MCC) is an essential component of the temporomandibular joint, which orchestrates the vertical growth of the mandibular ramus through endochondral ossification with distinctive modes of cell differentiation. Parathyroid hormone–related protein (PTHrP) is a master regulator of chondrogenesis; in the long bone epiphyseal growth plate, PTHrP expressed by resting zone chondrocytes promotes chondrocyte proliferation in the adjacent layer. However, how PTHrP regulates chondrogenesis in the MCC remains largely unclear. In this study, we used a Pthrp-mCherry knock-in reporter strain to map the localization of PTHrP+ cells in the MCC and define the function of PTHrP in the growing mandibular condyle. In the postnatal MCC of Pthrp mCherry/+ mice, PTHrP-mCherry was specifically expressed by cells in the superficial layer immediately adjacent to RUNX2-expressing cells in the polymorphic layer. PTHrP ligands diffused across the polymorphic and chondrocyte layers where its cognate receptor PTH1R was abundantly expressed. We further analyzed the mandibular condyle of Pthrp mCherry/mCherry mice lacking functional PTHrP protein (PTHrP-KO). At embryonic day (E) 18.5, the condylar process and MCC were significantly truncated in the PTHrP-KO mandible, which was associated with a significant reduction in cell proliferation across the polymorphic layer and a loss of SOX9+ cells in the chondrocyte layers. The PTHrP-KO MCC showed a transient increase in the number of Col10a1+ hypertrophic chondrocytes at E15.5, followed by a significant loss of these cells at E18.5, indicating that superficial layer–derived PTHrP prevents premature chondrocyte exhaustion in the MCC. The expression of Runx2, but not Sp7, was significantly reduced in the polymorphic layer of the PTHrP-KO MCC. Therefore, PTHrP released from cells in the superficial layer directly acts on cells in the polymorphic layer to promote proliferation of chondrocyte precursor cells and prevent their premature differentiation by maintaining Runx2 expression, revealing a unique PTHrP gradient-directed mechanism that regulates MCC chondrogenesis.

Enzymatic Synthesis of Aptamer-Polynucleotide Nanoparticles with High Anticancer Drug Loading for Targeted Delivery.

We report a targeted prodrug delivery platform that can deliver a cytostatic nucleobase analog with high drug loading. We chose fluorouracil (5FU), a drug used to treat various cancers, whose active metabolite 5-fluorodeoxyuridine monophosphate (5-FdUMP) is the antineoplastic agent. We use terminal deoxynucleotidyl transferase (TdT) to polymerize 5-fluorodeoxyuridine triphosphate (5-FdUTP) onto the 3'-end of an aptamer. We find that (i) addition of hydrophobic, unnatural nucleotides at the 3'-end of the 5-FdU polynucleotide by TdT leads to their spontaneous self-assembly into nuclease resistant micelles, (ii) aptamers presented on the micelle corona retain specificity for their cognate receptor on tumor cells, and (iii) the micelles deliver 5FU to tumor cells and exhibit greater cytotoxicity than the free drug. The modular design of our platform, consisting of a targeting moiety, a polynucleotide drug, and a self-assembly domain, can be adapted to encompass a range of polymerizable therapeutic nucleotides and targeting units.