Memory T-Cells Contribute to Calcium Release from Bones during Lactation in Mice.

Objective: Milk production during lactation places a high demand for calcium that is fulfilled both from maternal bone resorption and diet. While it is known that mammary gland-derived PTHrP drives bone resorption during lactation, the impact of postpartum estrogen loss on bone has been unclear. Methods: We used a case-control study design to test the effect of estrogen loss in lactating mice. Results: In the present study, we show for the first time that estrogen loss during lactation activates memory T-cells (TM) to produce TNFα and IL-17A to aid in bone resorption and calcium release. Our studies reveal a new mechanism for the release of calcium from bone postpartum. The findings provide several new insights. First, the immune system plays a critical role in milk production postpartum. Second, evolutionarily, the pathway serves the physiological purpose of increasing bone resorption to release calcium for breastmilk production postpartum but becomes maladaptive postmenopause, leading to osteoporosis. Finally, these results highlight the crosstalk between the brain-bone-breast-endocrine axis and the immune system during lactation.

Intermittent mechanical loading on mouse tibia accelerates longitudinal bone growth by inducing PTHrP expression in the female tibial growth plate.

It is not clear as to whether weight bearing and ambulation may affect bone growth. Our goal was to study the role of mechanical loading (one of the components of ambulation) on endochondral ossification and longitudinal bone growth. Thus, we applied cyclical, biologically relevant strains for a prolonged time period (4 weeks) to one tibia of juvenile mice, while using the contralateral one as an internal control. By the end of the 4-week loading period, the mean tibial growth of the loaded tibiae was significantly greater than that of the unloaded tibiae. The mean height and the mean area of the loaded tibial growth plates were greater than those of the unloaded tibiae. In addition, in female mice we found a greater expression of PTHrP in the loaded tibial growth plates than in the unloaded ones. Lastly, microCT analysis revealed no difference between loaded and unloaded tibiae with respect to the fraction of bone volume relative to the total volume of the region of interest or the tibial trabecular bone volume. Thus, our findings suggest that intermittent compressive forces applied on tibiae at mild-moderate strain magnitude induce a significant and persistent longitudinal bone growth. PTHrP expressed in the growth plate appears to be one growth factor responsible for stimulating endochondral ossification and bone growth in female mice.

PTHrP participates in the bone destruction of middle ear cholesteatoma via promoting macrophage differentiation into osteoclasts induced by RANKL. / PTHrP促进RANKLè¯±å¯¼å·¨å™¬ç»†èƒžåˆ†åŒ–ä¸ºç ´éª¨ç»†èƒžå‚ä¸Žä¸­è€³èƒ†è„‚ç˜¤éª¨ç ´å.

OBJECTIVES: Progressive bone resorption and destruction is one of the most critical clinical features of middle ear cholesteatoma, potentially leading to various intracranial and extracranial complications. However, the mechanisms underlying bone destruction in middle ear cholesteatoma remain unclear. This study aims to explore the role of parathyroid hormone-related protein (PTHrP) in bone destruction associated with middle ear cholesteatoma. METHODS: A total of 25 cholesteatoma specimens and 13 normal external auditory canal skin specimens were collected from patients with acquired middle ear cholesteatoma. Immunohistochemical staining was used to detect the expressions of PTHrP, receptor activator for nuclear factor-kappa B ligand (RANKL), and osteoprotegerin (OPG) in cholesteatoma and normal tissues. Tartrate-resistant acid phosphatase (TRAP) staining was used to detect the presence of TRAP positive multi-nucleated macrophages in cholesteatoma and normal tissues. Mono-nuclear macrophage RAW264.7 cells were subjected to interventions, divided into a RANKL intervention group and a PTHrP+ RANKL co-intervention group. TRAP staining was used to detect osteoclast formation in the 2 groups. The mRNA expression levels of osteoclast-related genes, including TRAP, cathepsin K (CTSK), and nuclear factor of activated T cell cytoplasmic 1 (NFATc1), were measured using real-time polymerase chain reaction (real-time PCR) after the interventions. Bone resorption function of osteoclasts was assessed using a bone resorption pit analysis. RESULTS: Immunohistochemical staining showed significantly increased expression of PTHrP and RANKL and decreased expression of OPG in cholesteatoma tissues (all P<0.05). PTHrP expression was significantly positively correlated with RANKL, the RANKL/OPG ratio, and negatively correlated with OPG expression (r=0.385, r=0.417, r=-0.316, all P<0.05). Additionally, the expression levels of PTHrP and RANKL were significantly positively correlated with the degree of bone destruction in cholesteatoma (r=0.413, r=0.505, both P<0.05). TRAP staining revealed a large number of TRAP-positive cells, including multi-nucleated osteoclasts with three or more nuclei, in the stroma surrounding the cholesteatoma epithelium. After 5 days of RANKL or PTHrP+RANKL co-intervention, the number of osteoclasts was significantly greater in the PTHrP+RANKL co-intervention group than that in the RANKL group (P<0.05), with increased mRNA expression levels of TRAP, CTSK, and NFATc1 (all P<0.05). Scanning electron microscopy of bone resorption pits showed that the number (P<0.05) and size of bone resorption pits on bone slices were significantly greater in the PTHrP+RANKL co-intervention group compared with the RANKL group. CONCLUSIONS: PTHrP may promote the differentiation of macrophages in the surrounding stroma of cholesteatoma into osteoclasts through RANKL induction, contributing to bone destruction in middle ear cholesteatoma.

Abnormal dental follicle cells: A crucial determinant in tooth eruption disorders (Review).

The dental follicle (DF) plays an indispensable role in tooth eruption by regulating bone remodeling through their influence on osteoblast and osteoclast activity. The process of tooth eruption involves a series of intricate regulatory mechanisms and signaling pathways. Disruption of the parathyroid hormone­related protein (PTHrP) in the PTHrP­PTHrP receptor signaling pathway inhibits osteoclast differentiation by DF cells (DFCs), thus resulting in obstructed tooth eruption. Furthermore, parathyroid hormone receptor­1 mutations are linked to primary tooth eruption failure. Additionally, the Wnt/ß­catenin, TGF­ß, bone morphogenetic protein and Hedgehog signaling pathways have crucial roles in DFC involvement in tooth eruption. DFC signal loss or alteration inhibits osteoclast differentiation, affects osteoblast and cementoblast differentiation, and suppresses DFC proliferation, thus resulting in failed tooth eruptions. Abnormal tooth eruption is also associated with a range of systemic syndromes and genetic diseases, predominantly resulting from pathogenic gene mutations. Among these conditions, the following disorders arise due to genetic mutations that disrupt DFCs and impede proper tooth eruption: Cleidocranial dysplasia associated with Runt­related gene 2 gene mutations; osteosclerosis caused by CLCN7 gene mutations; mucopolysaccharidosis type VI resulting from arylsulfatase B gene mutations; enamel renal syndrome due to FAM20A gene mutations; and dentin dysplasia caused by mutations in the VPS4B gene. In addition, regional odontodysplasia and multiple calcific hyperplastic DFs are involved in tooth eruption failure; however, they are not related to gene mutations. The specific mechanism for this effect requires further investigation. To the best of our knowledge, previous reviews have not comprehensively summarized the syndromes associated with DF abnormalities manifesting as abnormal tooth eruption. Therefore, the present review aims to consolidate the current knowledge on DFC signaling pathways implicated in abnormal tooth eruption, and their association with disorders of tooth eruption in genetic diseases and syndromes, thereby providing a valuable reference for future related research.

Astragalus Extract Mixture HT042 Alleviates Dexamethasone-Induced Bone Growth Retardation in Rat Metatarsal Bones.

The most widely used synthetic glucocorticoid, dexamethasone (DEX), causes stunted growth in children when used excessively or for long periods of time; however, there are still plenty of pediatric patients require long-term treatment with DEX. As an alternative, growth hormone is used in combination, but it has side effects, a high cost, and psychological factors, and it is not satisfactory in terms of effectiveness. It is necessary to develop a safe and affordable treatment that can replace it. The Korean Food and Drug Administration approved HT042, a standardized functional food ingredient, with the claim that it can help height growth of children. In this study, it was found that HT042 activated the Indian hedgehog/parathyroid hormone-related protein signaling pathway and enhanced the number of growth hormone receptors and insulin-like growth factor-1 receptors on the growth plate surface, which were reduced by DEX treatment, and restored growth retardation. In metatarsal bone and primary chondrocyte models, it was found that HT042 can promote the length of growth plate and recover DEX-induced growth retardation. It was also found that HT042 promotes cell proliferation using bromodeoxyuridine and terminal deoxynucleotidyl transferase dUTP nick end labeling assays; moreover, we verified increased expression of GHR/IGF-1R and Ihh/PTHrP pathway activity using qRT-PCR, western blotting, and siRNA analyses to verify its direct action on the growth plate. The anti-apoptotic effect of HT042 was identified by regulating the expression of apoptotic factors such as caspase-3, Bcl2, Bclx, and Bax. These results were identified using both ex vivo and in vitro models. Our study verified that co-administration of HT042 could recover the DEX induced growth retardation.

Design and development of in-vitro co-culture device for studying cellular crosstalk in varied tissue microenvironment.

Despite of being in different microenvironment, breast cancer cells influence the bone cells and persuade cancer metastasis from breast to bone. Multiple co-culture approaches have been explored to study paracrine signaling between these cells and to study the progression of cancer. However, lack of native tissue microenvironment remains a major bottleneck in existing co-culture technologies. Therefore, in the present study, a tumorigenic and an osteogenic microenvironment have been sutured together to create a multi-cellular environment and has been appraised to study cancer progression in bone tissue. The PCL-polystyrene and PCL-collagen fibrous scaffolds were characterized for tumorigenic and osteogenic potential induction on MDA-MB-231 and MC3T3-E1 cells respectively. Diffusion ability of crystal violet, glucose, and bovine serum albumin across the membrane were used to access the potential paracrine interaction facilitated by device. While in co-cultured condition, MDA-MB-231 cells showed EMT phenotype along with secretion of TNFα and PTHrP which lower down the expression of osteogenic markers including alkaline phosphatase, RUNX2, Osteocalcin and Osteoprotegerin. The cancer progression in bone microenvironment demonstrated the role and necessity of creating multiple tissue microenvironment and its contribution in studying multicellular disease progression and therapeutics.

Connexin hemichannels drive lactation-induced osteocyte acidification and perilacunar-canalicular remodeling.

The maternal skeleton experiences significant bone loss during lactation, followed by rapid restoration post weaning. Parathyroid-related protein (PTHrP)-induced acidification of the perilacunar matrix by osteocytes is crucial in this process, yet its mechanism remains unclear. Here, we identify Cx43 hemichannels (HCs) as key mediators of osteocyte acidification and perilacunar-canalicular remodeling (PLR). Utilizing transgenic mouse models expressing dominant-negative Cx43 mutants, we show that mice with impaired Cx43 HCs exhibit attenuated lactation-induced responses compared to wild-type and only gap junction-impaired groups, including lacunar enlargement, upregulation of PLR genes, and bone loss with compromised mechanical properties. Furthermore, inhibition of HCs by a Cx43 antibody blunts PTHrP-induced calcium influx and protein kinase A activation, followed by impaired osteocyte acidification. Additionally, impeded HCs suppress bone recovery during the post-lactation period. Our findings highlight the pivotal role of Cx43 HCs in orchestrating dynamic bone changes during lactation and recovery by regulating acidification and remodeling enzyme expression.

A mathematical model of signalling molecule-mediated processes during regeneration of osteochondral defects after chondrocyte implantation.

Treating bone-cartilage defects is a fundamental clinical problem. The ability of damaged cartilage to self-repair is limited due to its avascularity. Left untreated, these defects can lead to osteoarthritis. Details of osteochondral defect repair are elusive, but animal models indicate healing occurs via an endochondral ossification-like process, similar to that in the growth plate. In the growth plate, the signalling molecules parathyroid hormone-related protein (PTHrP) and Indian Hedgehog (Ihh) form a feedback loop regulating chondrocyte hypertrophy, with Ihh inducing and PTHrP suppressing hypertrophy. To better understand this repair process and to explore the regulatory role of signalling molecules on the regeneration process, we formulate a reaction-diffusion mathematical model of osteochondral defect regeneration after chondrocyte implantation. The drivers of healing are assumed to be chondrocytes and osteoblasts, and their interaction via signalling molecules. We model cell proliferation, migration and chondrocyte hypertrophy, and matrix production and conversion, spatially and temporally. We further model nutrient and signalling molecule diffusion and their interaction with the cells. We consider the PTHrP-Ihh feedback loop as the backbone mechanisms but the model is flexible to incorporate extra signalling mechanisms if needed. Our mathematical model is able to represent repair of osteochondral defects, starting with cartilage formation throughout the defect. This is followed by chondrocyte hypertrophy, matrix calcification and bone formation deep inside the defect, while cartilage at the surface is maintained and eventually separated from the deeper bone by a thin layer of calcified cartilage. The complete process requires around 48 months. A key highlight of the model demonstrates that the PTHrP-Ihh loop alone is insufficient and an extra mechanism is required to initiate chondrocyte hypertrophy, represented by a critical cartilage density. A parameter sensitivity study reveals that the timing of the repair process crucially depends on parameters, such as the critical cartilage density, and those describing the actions of PTHrP to suppress hypertrophy, such as its diffusion coefficient, threshold concentration and degradation rate.

Ex vivo organotypic bone slice culture reveals preferential chondrogenesis after sustained growth plate injury.

Postnatal bone growth primarily relies on chondrocyte proliferation and osteogenic differentiation within the growth plate (GP) via endochondral ossification. Despite its importance, the GP is vulnerable to injuries, affecting 15-30 % of bone fractures. These injuries may lead to growth discrepancies, influence bone length and shape, and negatively affecting the patient's quality of life. This study aimed to investigate the molecular and cellular physiological and pathophysiological regeneration following sustained growth plate injury (GPI) in an ex vivo rat femur organotypic culture (OTC) model. Specifically, focusing on postnatal endochondral ossification process. 300 µm thick ex vivo bone cultures with a 2 mm long horizontal GPI was utilized. After 15 days of cultivation, gene expression analysis, histological and immunohistochemistry staining's were conducted to analyze key markers of endochondral ossification. In our OTCs we observed a significant increase in Sox9 expression due to GPI at day 15. The Ihh-PTHrP feedback loop was affected, favoring chondrocyte proliferation and maturation. Ihh levels increased significantly on day 7 and day 15, while PTHrP was downregulated on day 7. GPI had no impact on osteoclast number and activity, but gene expression analysis indicated OTCs' efforts to inhibit osteoclast differentiation and activation, thereby reducing bone resorption. In conclusion, our study provides novel insights into the molecular and cellular mechanisms underlying postnatal bone growth and regeneration following growth plate injury (GPI). We demonstrate that chondrocyte proliferation and differentiation play pivotal roles in the regeneration process, with the Ihh-PTHrP feedback loop modulating these processes. Importantly, our ex vivo rat femur organotypic culture model allows for the detailed investigation of these processes, providing a valuable tool for future research in the field of skeletal biology and regenerative medicine.

Interrogating Estrogen Signaling Pathways in Human ER-Positive Breast Cancer Cells Forming Bone Metastases in Mice.

Breast cancer bone metastases (BMET) are incurable, primarily osteolytic, and occur most commonly in estrogen receptor-α positive (ER+) breast cancer. ER+ human breast cancer BMET modeling in mice has demonstrated an estrogen (E2)-dependent increase in tumor-associated osteolysis and bone-resorbing osteoclasts, independent of estrogenic effects on tumor proliferation or bone turnover, suggesting a possible mechanistic link between tumoral ERα-driven osteolysis and ER+ bone progression. To explore this question, inducible secretion of the osteolytic factor, parathyroid hormone-related protein (PTHrP), was utilized as an in vitro screening bioassay to query the osteolytic potential of estrogen receptor- and signaling pathway-specific ligands in BMET-forming ER+ human breast cancer cells expressing ERα, ERß, and G protein-coupled ER. After identifying genomic ERα signaling, also responsibility for estrogen's proliferative effects, as necessary and sufficient for osteolytic PTHrP secretion, in vivo effects of a genomic-only ER agonist, estetrol (E4), on osteolytic ER+ BMET progression were examined. Surprisingly, while pharmacologic effects of E4 on estrogen-dependent tissues, including bone, were evident, E4 did not support osteolytic BMET progression (vs robust E2 effects), suggesting an important role for nongenomic ER signaling in ER+ metastatic progression at this site. Because bone effects of E4 did not completely recapitulate those of E2, the relative importance of nongenomic ER signaling in tumor vs bone cannot be ascertained here. Nonetheless, these intriguing findings suggest that targeted manipulation of estrogen signaling to mitigate ER+ metastatic progression in bone may require a nuanced approach, considering genomic and nongenomic effects of ER signaling on both sides of the tumor/bone interface.

The Impact of Non-bone Metastatic Cancer on Musculoskeletal Health.

PURPOSE OF REVIEW: The purpose of this review is to discuss the musculoskeletal consequences of cancer, including those that occur in the absence of bone metastases. RECENT FINDINGS: Cancer patients frequently develop cachexia, a debilitating condition reflected by weight loss and skeletal muscle wasting. The negative effects that tumors exert on bone health represents a growing interest amongst cachexia researchers. Recent clinical and pre-clinical evidence demonstrates cancer-induced bone loss, even in the absence of skeletal metastases. Together with muscle wasting, losses in bone demonstrates the impact of cancer on the musculoskeletal system. Identifying therapeutic targets that comprehensively protect musculoskeletal health is essential to improve the quality of life in cancer patients and survivors. IL-6, RANKL, PTHrP, sclerostin, and TGF-ß superfamily members represent potential targets to counteract cachexia. However, more research is needed to determine the efficacy of these targets in protecting both skeletal muscle and bone.

Parathyroid hormone related-protein (PTHrP) in tissues with poor prognosis in prostate cancer patients.

BACKGROUND: Parathyroid hormone-related peptide (PTHrP) is known to have a pivotal role in the progression of various solid tumors, among which prostate cancer stands out. However, the extent of PTHrP expression and its clinical implications in prostate cancer patients remain shrouded in obscurity. The primary objective of this research endeavor was to shed light on the relevance of PTHrP in the context of prostate cancer patients and to uncover the potential underlying mechanisms. METHODS: The expression of PTHrP, E-cadherin, and vimentin in tumor tissues of 88 prostate cancer patients was evaluated by immunohistochemical technique. Subsequently, the associations between PTHrP and clinicopathological parameters and prognosis of patients with prostate cancer were analyzed. RESULTS: Immunohistochemical analysis showed that the expression rates of PTHrP, E-cadherin, and vimentin in prostate cancer tissues were 95.5%, 88.6%, and 84.1%, respectively. Patients with a high level of PTHrP had a decreased expression of E-cadherin (P = .013) and an increased expression of vimentin (P = .010) compared with patients with a low level of PTHrP. Besides, the high expression of PTHrP was significantly correlated with a higher level of initial prostate-specific antigen (P = .026), positive lymph node metastasis (P = .010), osseous metastasis (P = .004), and Gleason score (P = .026). Moreover, patients with a high level of PTHrP had shorter progression-free survival (P = .002) than patients with a low level of PTHrP. CONCLUSION: The present study indicates that PTHrP is associated with risk factors of poor outcomes in prostate cancer, while epithelial-mesenchymal transition may be involved in this process.

PTHrP attenuates spontaneous contractions in detrusor smooth muscle of the rat bladder by activating spontaneous transient outward potassium currents.

Parathyroid hormone-related protein (PTHrP) released from detrusor smooth muscle (DSM) cells upon bladder distension attenuates spontaneous phasic contractions (SPCs) in DSM and associated afferent firing to facilitate urine storage. Here, we investigate the mechanisms underlying PTHrP-induced inhibition of SPCs, focusing on large-conductance Ca2+-activated K+ channels (BK channels) that play a central role in stabilizing DSM excitability. Perforated patch-clamp techniques were applied to DSM cells of the rat bladder dispersed using collagenase. Isometric tension changes were recorded from DSM strips, while intracellular Ca2+ dynamics were visualized using Cal520 AM -loaded DSM bundles. DSM cells developed spontaneous transient outward potassium currents (STOCs) arising from the opening of BK channels. PTHrP (10 nM) increased the frequency of STOCs without affecting their amplitude at a holding potential of - 30 mV but not - 40 mV. PTHrP enlarged depolarization-induced, BK-mediated outward currents at membrane potentials positive to + 20 mV in a manner sensitive to iberiotoxin (100 nM), the BK channel blocker. The PTHrP-induced increases in BK currents were also prevented by inhibitors of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) (CPA 10 µM), L-type voltage-dependent Ca2+ channel (LVDCC) (nifedipine 3 µM) or adenylyl cyclase (SQ22536 100 µM). PTHrP had no effect on depolarization-induced LVDCC currents. PTHrP suppressed and slowed SPCs in an iberiotoxin (100 nM)-sensitive manner. PTHrP also reduced the number of Ca2+ spikes during each burst of spontaneous Ca2+ transients. In conclusion, PTHrP accelerates STOCs discharge presumably by facilitating SR Ca2+ release which prematurely terminates Ca2+ transient bursts resulting in the attenuation of SPCs.

PTHrP intracrine actions divergently influence breast cancer growth through p27 and LIFR.

The role of parathyroid hormone (PTH)-related protein (PTHrP) in breast cancer remains controversial, with reports of PTHrP inhibiting or promoting primary tumor growth in preclinical studies. Here, we provide insight into these conflicting findings by assessing the role of specific biological domains of PTHrP in tumor progression through stable expression of PTHrP (-36-139aa) or truncated forms with deletion of the nuclear localization sequence (NLS) alone or in combination with the C-terminus. Although the full-length PTHrP molecule (-36-139aa) did not alter tumorigenesis, PTHrP lacking the NLS alone accelerated primary tumor growth by downregulating p27, while PTHrP lacking the NLS and C-terminus repressed tumor growth through p27 induction driven by the tumor suppressor leukemia inhibitory factor receptor (LIFR). Induction of p27 by PTHrP lacking the NLS and C-terminus persisted in bone disseminated cells, but did not prevent metastatic outgrowth, in contrast to the primary tumor site. These data suggest that the PTHrP NLS functions as a tumor suppressor, while the PTHrP C-terminus may act as an oncogenic switch to promote tumor progression through differential regulation of p27 signaling.

Exploring dual effects of dinutuximab beta on cell death and proliferation of insulinoma.

Insulinoma INS-1 cells are pancreatic beta cell tumors. Dinutuximab beta (DB) is a monoclonal antibody used in the treatment of neuroblastoma. The aim of this study is to investigate the effects of DB on pancreatic beta cell tumors at the molecular level. DB (Qarziba®) was available from EUSA Pharma. Streptozotocin (STZ) was used induce to cell cytotoxicity. DB was applied to the cells before or after the STZ application. KCND3, KCNN4, KCNK1, and PTHrP gene expression levels were analyzed by q-RT-PCR, and protein levels were analyzed by Western blotting. Analysis of glucose-stimulated insulin secretion was performed. Ca+2 and CA19-9 levels were determined by the ELISA kit. PERK, CHOP, HSP90, p-c-Jun, p-Atf2, and p-Elk1 protein levels were analyzed by simple WES. Decreased KCND3, KCNK1, and PTHrP protein levels and increased KCND3, KCNN4, KCNK1, and PTHrP gene expression levels were observed with DB applied after STZ application. Cell dysfunction was detected with DB applied before and after STZ application. Ca19-9 and Ca+2 levels were increased with DB applied after STZ application. PERK, CHOP, and p-Elk1 levels decreased, while HSP90 levels increased with DB applied after STZ application. CHOP, p-Akt-2, and p-c-Jun levels increased in the DB group. As a result, INS-1 cells go to cell death via the ERK signaling pathway without ER stress and release insulin with the decrease of K+ channels and an increase in Ca+2 levels with DB applied after STZ application. Moreover, the cells proliferate via JNK signaling with DB application. DB holds promise for the treatment of insulinoma. The study should be supported by in vivo studies.

Hedgehog activation promotes osteogenic fates of growth plate resting zone chondrocytes through transient clonal competency.

The resting zone of the postnatal growth plate is organized by slow-cycling chondrocytes expressing parathyroid hormone-related protein (PTHrP), which include a subgroup of skeletal stem cells that contribute to the formation of columnar chondrocytes. The PTHrP-Indian hedgehog feedback regulation is essential for sustaining growth plate activities; however, molecular mechanisms regulating cell fates of PTHrP+ resting chondrocytes and their eventual transformation into osteoblasts remain largely undefined. Here, in a mouse model, we specifically activated Hedgehog signaling in PTHrP+ resting chondrocytes and traced the fate of their descendants using a tamoxifen-inducible Pthrp-creER line with patched-1-floxed and tdTomato reporter alleles. Hedgehog-activated PTHrP+ chondrocytes formed large, concentric, clonally expanded cell populations within the resting zone ("patched roses") and generated significantly wider columns of chondrocytes, resulting in hyperplasia of the growth plate. Interestingly, Hedgehog-activated PTHrP+ cell descendants migrated away from the growth plate and transformed into trabecular osteoblasts in the diaphyseal marrow space in the long term. Therefore, Hedgehog activation drives resting zone chondrocytes into transit-amplifying states as proliferating chondrocytes and eventually converts these cells into osteoblasts, unraveling a potentially novel Hedgehog-mediated mechanism that facilitates osteogenic cell fates of PTHrP+ skeletal stem cells.

Local injection of abaloparatide promotes mandibular condyle lengthening in adolescent rats via enhancing chondrogenesis and ossification.

BACKGROUND: Mandibular condylar hypoplasia negatively affects patient's facial appearance and dentofacial function. OBJECTIVE: To investigate the effect of local injection of the drug abaloparatide (ABL), an analogue of parathyroid hormone related protein (PTHrP), on promoting lengthening of the mandibular condyle. METHODS: Thirty adolescent male Sprague-Dawley rats were randomly divided into two groups, which received the injection of ABL or normal saline (the control) every 3 days in the temporomandibular joint (TMJ) cavity. Cone-beam computed tomography and immunohistochemistry assays were performed at 2, 4 and 6 weeks since the injection. Mandibular condylar chondrocytes (MCC) and pre-osteoblasts were treated with ABL or PBS, followed by the CCK-8 detection, IC50, real-time PCR assay, Western Blot and immunofluorescence staining. RESULTS: In vivo, compared with the control, the ABL group significantly increased the mandibular condylar process length (by 1.34 ± 0.59 mm at 6 weeks), the thickness of the cartilage layer, and enhanced the matrix synthesis. The ABL group had significant up-regulation of SOX 9, COL II, PTHrP and PTH1R, down-regulation of COL X in the cartilage, up-regulation of RUNX 2, and unchanged osteoclastogenesis in the subchondral bone. In vitro, the intra-TMJ injection of ABL promoted the MCC proliferation, with up-regulated expression of chondrogenic genes, and enhanced osteogenic differentiation of the pre-osteoblasts. CONCLUSIONS: Intra-TMJ injection of abaloparatide promotes mandibular condyle lengthening in the adolescent rats via enhancing chondrogenesis in the mandibular condylar cartilage and ossification in the subchondral bone.

Comparative study in estrogen-depleted mice identifies skeletal and osteocyte transcriptomic responses to abaloparatide and teriparatide.

Osteocytes express parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptors and respond to the PTHrP analog abaloparatide (ABL) and to the PTH 1-34 fragment teriparatide (TPTD), which are used to treat osteoporosis. Several studies indicate overlapping but distinct skeletal responses to ABL or TPTD, but their effects on cortical bone may differ. Little is known about their differential effects on osteocytes. We compared cortical osteocyte and skeletal responses to ABL and TPTD in sham-operated and ovariectomized mice. Administered 7 weeks after ovariectomy for 4 weeks at a dose of 40 µg/kg/d, TPTD and ABL had similar effects on trabecular bone, but ABL showed stronger effects in cortical bone. In cortical osteocytes, both treatments decreased lacunar area, reflecting altered peri-lacunar remodeling favoring matrix accumulation. Osteocyte RNA-Seq revealed that several genes and pathways were altered by ovariectomy and affected similarly by TPTD and ABL. Notwithstanding, several signaling pathways were uniquely regulated by ABL. Thus, in mice, TPTD and ABL induced a positive osteocyte peri-lacunar remodeling balance, but ABL induced stronger cortical responses and affected the osteocyte transcriptome differently. We concluded that ABL affected the cortical osteocyte transcriptome in a manner subtly different from TPTD, resulting in more beneficial remodeling/modeling changes and homeostasis of the cortex.

Cranial Neural Crest Specific Deletion of Alpl (TNAP) via P0-Cre Causes Abnormal Chondrocyte Maturation and Deficient Cranial Base Growth.

Bone growth plate abnormalities and skull shape defects are seen in hypophosphatasia, a heritable disorder in humans that occurs due to the deficiency of tissue nonspecific alkaline phosphatase (TNAP, Alpl) enzyme activity. The abnormal development of the cranial base growth plates (synchondroses) and abnormal skull shapes have also been demonstrated in global Alpl-/- mice. To distinguish local vs. systemic effects of TNAP on skull development, we utilized P0-Cre to knockout Alpl only in cranial neural crest-derived tissues using Alpl flox mice. Here, we show that Alpl deficiency using P0-Cre in cranial neural crest leads to skull shape defects and the deficient growth of the intersphenoid synchondrosis (ISS). ISS chondrocyte abnormalities included increased proliferation in resting and proliferative zones with decreased apoptosis in hypertrophic zones. ColX expression was increased, which is indicative of premature differentiation in the absence of Alpl. Sox9 expression was increased in both the resting and prehypertrophic zones of mutant mice. The expression of Parathyroid hormone related protein (PTHrP) and Indian hedgehog homolog (IHH) were also increased. Finally, cranial base organ culture revealed that inorganic phosphate (Pi) and pyrophosphate (PPi) have specific effects on cell signaling and phenotype changes in the ISS. Together, these results demonstrate that the TNAP expression downstream of Alpl in growth plate chondrocytes is essential for normal development, and that the mechanism likely involves Sox9, PTHrP, IHH and PPi.

CaSR modulates proliferation of the superficial zone cells in temporomandibular joint cartilage via the PTHrP nuclear localization sequence.

The superficial zone cells in mandibular condylar cartilage are proliferative. The present purpose was to delineate the relation of calcium-sensing receptor (CaSR) and parathyroid hormone-related peptide nuclear localization sequence (PTHrP87-139 ), and their role in the proliferation behaviors of the superficial zone cells. A gain- and loss-of-function strategy were used in an in vitro fluid flow shear stress (FFSS) model and an in vivo bilateral elevation bite model which showed mandibular condylar cartilage thickening. CaSR and PTHrP87-139 were modulated through treating the isolated superficial zone cells with activator/SiRNA and via deleting CaSR or parathyroid hormone-related peptide (PTHrP) gene in mice with the promoter gene of proteoglycan 4 (Prg4-CreERT2 ) in the tamoxifen-inducible pattern with or without additional injection of Cinacalcet, the CaSR agonist, or PTHrP87-139 peptide. FFSS stimulated CaSR and PTHrP expression, and accelerated proliferation of the Prg4-expressing superficial zone cells, in which process CaSR acted as an up-streamer of PTHrP. Proteoglycan 4 specific knockout of CaSR or PTHrP reduced the cartilage thickness, suppressed the proliferation and early differentiation of the superficial zone cells, and inhibited cartilage thickening and matrix production promoted by bilateral elevation bite. Injections of CaSR agonist Cinacalcet could not improve the phenotype caused by PTHrP mutation. Injections of PTHrP87-139 peptide rescued the cartilage from knockout of CaSR gene. CaSR modulates proliferation of the superficial zone cells in mandibular condylar cartilage through activation of PTHrP nuclear localization sequence. Our data support the therapeutic target of CaSR in promoting PTHrP production in superficial zone cartilage.