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Methylphenidate Promotes Premature Growth Plate Closure: In Vitro Evidence.

ABSTRACT: It is well known that patients with attention deficit hyperactivity disorder treated with stimulants, such as methylphenidate hydrochloride (MPH), have reduced height and weight. Even though MPH has an anorexigenic effect, an additional impact of this drug on the growth plate cannot be discarded. In this study, we aimed to determine the cellular effect of MPH on an in vitro growth plate model. We tested the effects of MPH on the viability and proliferation of a prechondrogenic cell line via an MTT assay. In vitro differentiation of this cell line was performed, and cell differentiation was evaluated through the expression of cartilage- and bone-related genes as measured via RT-PCR. MPH did not alter the viability or proliferation of prechondrogenic cells. However, it reduced the expression of cartilage extracellular matrix-related genes (type II collagen and aggrecan) and increased the expression of genes involved in growth plate calcification (Runx2, type I collagen, and osteocalcin) at different phases of their differentiation process. Our results evidence that MPH upregulates genes associated with growth plate hypertrophic differentiation. This may induce premature closure of the growth plate, which would contribute to the growth retardation that has been described to be induced by this drug.

SUBMITTER: Pazos-Perez A

PROVIDER: S-EPMC9968202 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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Methylphenidate Promotes Premature Growth Plate Closure: In Vitro Evidence.

Pazos-Pérez Andrés A Piñeiro-Ramil María M Franco-Trepat Eloi E Guillán-Fresco María M López-López Verónica V Jorge-Mora Alberto A Alonso-Pérez Ana A Gómez Rodolfo R

International journal of molecular sciences 20230220 4

It is well known that patients with attention deficit hyperactivity disorder treated with stimulants, such as methylphenidate hydrochloride (MPH), have reduced height and weight. Even though MPH has an anorexigenic effect, an additional impact of this drug on the growth plate cannot be discarded. In this study, we aimed to determine the cellular effect of MPH on an in vitro growth plate model. We tested the effects of MPH on the viability and proliferation of a prechondrogenic cell line via an M ...[more]

PMID: 36835608

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Project description:The growth plates are key engines of skeletal development and growth and contain a top reserve zone followed by maturation zones of proliferating, prehypertrophic, and hypertrophic/mineralizing chondrocytes. Trauma or drug treatment of certain disorders can derange the growth plates and cause accelerated maturation and premature closure, one example being anti-hedgehog drugs such as LDE225 (Sonidegib) used against pediatric brain malignancies. Here we tested whether such acceleration and closure in LDE225-treated mice could be prevented by co-administration of a selective retinoid antagonist, based on previous studies showing that retinoid antagonists can slow down chondrocyte maturation rates. Treatment of juvenile mice with an experimental dose of LDE225 for 2 days (100 mg/kg by gavage) initially caused a significant shortening of long bone growth plates, with concomitant decreases in chondrocyte proliferation; expression of Indian hedgehog, Sox9, and other key genes; and surprisingly, the number of reserve progenitors. Growth plate involution followed with time, leading to impaired long bone lengthening. Mechanistically, LDE225 treatment markedly decreased the expression of retinoid catabolic enzyme Cyp26b1 within growth plate, whereas it increased and broadened the expression of retinoid synthesizing enzyme Raldh3, thus subverting normal homeostatic retinoid circuitries and in turn accelerating maturation and closure. All such severe skeletal and molecular changes were prevented when LDE-treated mice were co-administered the selective retinoid antagonist CD2665 (1.5 mg/kg/d), a drug targeting retinoid acid receptor γ, which is most abundantly expressed in growth plate. When given alone, CD2665 elicited the expected maturation delay and growth plate expansion. In vitro data showed that LDE225 acted directly to dampen chondrogenic phenotypic expression, a response fully reversed by CD2665 co-treatment. In sum, our proof-of-principle data indicate that drug-induced premature growth plate closures can be prevented or delayed by targeting a separate phenotypic regulatory mechanism in chondrocytes. The translation applicability of the findings remains to be studied. © 2021 American Society for Bone and Mineral Research (ASBMR).

Project description:Prostaglandin E2 (PGE2) plays an important role in bone development and metabolism. To interfere therapeutically in the PGE2 pathway, however, knowledge about the involved enzymes (cyclooxygenases) and receptors (PGE2 receptors) is essential. We therefore examined the production of PGE2 in cultured growth plate chondrocytes in vitro and the effects of exogenously added PGE2 on cell proliferation. Furthermore, we analysed the expression and spatial distribution of cyclooxygenase (COX)-1 and COX-2 and PGE2 receptor types EP1, EP2, EP3 and EP4 in the growth plate in situ and in vitro. PGE2 synthesis was determined by mass spectrometry, cell proliferation by DNA [3H]-thymidine incorporation, mRNA expression of cyclooxygenases and EP receptors by RT-PCR on cultured cells and in homogenized growth plates. To determine cellular expression, frozen sections of rat tibial growth plate and primary chondrocyte cultures were stained using immunohistochemistry with polyclonal antibodies directed towards COX-1, COX-2, EP1, EP2, EP3, and EP4. Cultured growth plate chondrocytes transiently secreted PGE2 into the culture medium. Although both enzymes were expressed in chondrocytes in vitro and in vivo, it appears that mainly COX-2 contributed to PGE2-dependent proliferation. Exogenously added PGE2 stimulated DNA synthesis in a dose-dependent fashion and gave a bell-shaped curve with a maximum at 10-8 M. The EP1/EP3 specific agonist sulprostone and the EP1-selective agonist ONO-D1-004 increased DNA synthesis. The effect of PGE2 was suppressed by ONO-8711. The expression of EP1, EP2, EP3, and EP4 receptors in situ and in vitro was observed; EP2 was homogenously expressed in all zones of the growth plate in situ, whereas EP1 expression was inhomogenous, with spared cells in the reserve zone. In cultured cells these four receptors were expressed in a subset of cells only. The most intense staining for the EP1 receptor was found in polygonal cells surrounded by matrix. Expression of receptor protein for EP3 and EP4 was observed also in rat growth plates. In cultured chrondrocytes, however, only weak expression of EP3 and EP4 receptor was detected. We suggest that in growth plate chondrocytes, COX-2 is responsible for PGE2 release, which stimulates cell proliferation via the EP1 receptor.

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Methylphenidate Promotes Premature Growth Plate Closure: In Vitro Evidence.

Publications

Methylphenidate Promotes Premature Growth Plate Closure: In Vitro Evidence.

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