Pleckstrin homology (PH) domain and leucine rich repeat phosphatase 1 (Phlpp1) suppresses parathyroid hormone receptor 1 (Pth1r) expression and signaling during bone growth.

Abstract

A reader of the Journal of Bone and Mineral Research (JBMR®) has informed the authors that in the article by Weaver et al.,(1) the concentration of PTH(7–34) used in vivo was incorrectly stated in the figure legends. The errors are in legends to Figures 3–5, as well as Supplemental Figures 2, 5, and 6. The correct concentration appears in the Materials and Methods section and is 100 μg/kg body weight per day, not 100 mg/kg/day. The corrected figure legends appear below, and the authors apologize for these errors. Fig. 3 Daily administration of PTH(7–34) reverses short limb length in Phlpp1−/− mice. (A,C) Tibia and (B,D) femur lengths were evaluated in 4-week-old male WT or Phlpp1−/− mice given daily injections of PTH(7–34) (100 μg/kg body weight/day) or vehicle (0.1% BSA in PBS). The same treatments were administered to female WT and Phlpp1−/− mice and tibia (E,G) and femur (F,H) lengths were evaluated at 4 weeks of age. Scale bar = 5 mm. Statistically significant differences were determined with two-way ANOVA with Tukey's post-hoc test. Fig. 4 Phlpp1 and Pth1r coordinate to regulate growth plate development. (A,B) 3D renderings of proximal tibial growth plates were generated from μCT scans of 4-week-old WT or Phlpp1−/− male mice given daily injections of PTH(7–34) (100 μg/kg body weight/day) or vehicle. Scale bar on 2D capture = 1mm. Color scale bar is in mm. One representative 3D rendering is shown for each group. (C) Areas of proliferative and hypertrophic zones of the proximal tibia growth plate were quantified. (D) Safranin O/Fast Green staining was performed on the proximal tibial growth plate. Scale bar = 50 μm. Vertical black lines indicate either P = proliferative zone or H = hypertrophic zone. (E) In situ hybridization was performed for Col10a1. Scale bar = 50 μm. (F,G) WT or Phlpp1−/− mice were injected with either vehicle or PTH(7–34) daily as described above from postnatal day 1 (P1) through P5. On P5, BrdU was administered 2 hours prior to euthanasia. (F) Percentage of cells that were BrdU-positive in the proximal tibial growth plate was quantified, as represented in G. Scale bar in G = 50 μm. Statistically significant differences were determined with two-way ANOVA with Tukey's post hoc test. Fig. 5 Daily administration of PTH(7–34) does not affect bone mass. (A) 2D reconstructions from μCT of the distal femur of 4-week-old WT or Phlpp1−/− male mice given daily injections of PTH(7–34) (100 μg/kg body weight/day) or vehicle. Scale bar = 1 mm. Trabecular parameters analyzed via μCT were (B) bone volume/tissue volume, (C) trabecular number, and (D) bone mineral density. Cortical parameters included (E) tissue mineral density, (F) cortical thickness, (G) total tissue cross sectional area, and (H) cortical area fraction. Statistically significant differences were determined with two-way ANOVA with Tukey's post hoc test or Student's test as indicated. Supplementary Fig. S2 Low magnification images of the growth plate. 5× magnification images of (A) the growth plates of 4-week-old WT or Phlpp1−/−male mice after performing in situ hybridization (ISH) or immunohistochemistry (IHC) to detect Pth1r. Scale bar = 100 μm. (B,C) Four-week-old WT or Phlpp1−/− mice were given daily injections of PTH(7–34) (100 μg/kg body weight/day) or vehicle. The proximal tibial growth plate was (B) stained with Safranin O/Fast Green and (C) probed for Col10a1 using ISH. (D) WT or Phlpp1−/− mice were injected with either vehicle or PTH(7–34) daily as described above from postnatal day 1 (P1) through P5. On P5, BrdU labeling reagent was administered 2 hours prior to euthanasia and BrdU-positive cells were identified. Supplementary Fig. S5 Daily administration of PTH(7–34) has an intermediate effect on rescuing limb length in Phlpp1 heterozygotes compared to WT or Phlpp1−/− mice. (A,E) Femur length, (B,F) tibia length, (C,G) body length, and (D,H) body weight were evaluated in 4-week-old male and female WT (Phlpp1+/+), HET (Phlpp1+/−), or KO (Phlpp1−/−) mice given daily injections of PTH(7–34) (100 μg/kg body weight/day) or vehicle (0.1% BSA in PBS). Statistically significant differences were determined with two-way ANOVA with Tukey's post hoc test. Supplementary Fig. S6 Daily administration of PTH(7–34) reverses short body length and low body weight in Phlpp1−/−mice. (A,C) Tail-to-snout body length and (B,D) body weight were evaluated in 4-week-old male and female WT or Phlpp1−/− mice given daily injections of PTH(7–34) (100 μg/kg body weight/day) or vehicle (0.1% BSA in PBS). Statistically significant differences were determined with two-way ANOVA with Tukey's post hoc test. Reference 1. Weaver S, Taylor E, Zars E, Arnold K, Bradley E, Westendorf J. Pleckstrin homology (PH) domain and leucine rich repeat phosphatase 1 (Phlpp1) suppresses parathyroid hormone receptor 1 (Pth1r) expression and signaling during bone growth. J Bone Miner Res. 2021;36(5):986-989. https://doi.org/10.1002/jbmr.4248.