Conditional deletion of Pkd1 in osteocytes disrupts skeletal mechanosensing in mice

Abstract

We investigated whether polycystin-1 is a bone mechanosensor. We conditionally deleted Pkd1 in mature osteoblasts/osteocytes by crossing Dmp1-Cre with Pkd1(flox/m1Bei) mice, in which the m1Bei allele is nonfunctional. We assessed in wild-type and Pkd1-deficient mice the response to mechanical loading in vivo by ulna loading and ex vivo by measuring the response of isolated osteoblasts to fluid shear stress. We found that conditional Pkd1 heterozygotes (Dmp1-Cre;Pkd1(flox/+)) and null mice (Pkd1(Dmp1-cKO)) exhibited a ∼ 40 and ∼ 90% decrease, respectively, in functional Pkd1 transcripts in bone. Femoral bone mineral density (12 vs. 27%), trabecular bone volume (32 vs. 48%), and cortical thickness (6 vs. 17%) were reduced proportionate to the reduction of Pkd1 gene dose, as were mineral apposition rate (MAR) and expression of Runx2-II, Osteocalcin, Dmp1, and Phex. Anabolic load-induced periosteal lamellar MAR (0.58 ± 0.14; Pkd1(Dmp1-cKO) vs. 1.68 ± 0.34 μm/d; control) and increases in Cox-2, c-Jun, Wnt10b, Axin2, and Runx2-II gene expression were significantly attenuated in Pkd1(Dmp1-cKO) mice compared with controls. Application of fluid shear stress to immortalized osteoblasts from Pkd1(null/null) and Pkd1(m1Bei/m1Bei)-derived osteoblasts failed to elicit the increments in cytosolic calcium observed in wild-type controls. These data indicate that polycystin-1 is essential for the anabolic response to skeletal loading in osteoblasts/osteocytes.

Figure 1.

Dmp1-Cre-mediated conditional deletion of Pkd1 from the floxed Pkd1^flox allele in different tissues. A) Schematic illustration of wild-type Pkd1⁺, mutant Pkd1^m1Bei, and floxed Pkd1 allele before (Pkd1^flox) and after (Pkd1^Δflox) deletion of the lox P cassette containing exons 2–4 via Cre-mediated recombination. Slashes indicate all the introns and exons omitted between exons 5 and 25. B) Genotype PCR analysis of different tissues that were harvested from 16-wk-old Dmp1-Cre;Pkd1^flox/m1Bei mice. Pkd1 Beier and floxed alleles existed in all tested tissues, including bone. However, Dmp1-Cre-mediated recombination of excised floxed Pkd1^Δflox allele occurred in bone tissues, such as calvarias and femur, but also had a leakage in the brain, muscle, and intestine. C) Real-time RT-PCR analysis of total Pkd1 transcripts. Expression of total Pkd1 transcripts was performed using Pkd1-allele-specific primers, as described in Materials and Methods. Normal Pkd1⁺ vs. cyclophilin A is normalized to the mean ratio of 5 control mice, set to 1. Percentage of conditional deleted and mutant transcripts was calculated from the relative levels of the normal Pkd1⁺ transcripts in different Pkd1 exons. Data are expressed as the percentage of wild-type Pkd1⁺ and Pkd1^flox, mutant Pkd1^m1Bei, and conditionally deleted Pkd1^Δflox allele expressions in Pkd1^flox/+ control and Dmp1-Cre;Pkd1^flox/− conditionally null mice. D) Histology of adult kidney. Hematoxylin-and-eosin (H&E)-stained sections from 6-wk-old mice failed to identify any cystic tubules in either cortical or medullary regions of kidney from Dmp1-Cre;Pkd1^flox/+ or Pkd1^Dmp1-cKO mice, consistent with the absence of Dmp1-Cre expression in the kidney. In contrast, ablation of Pkd1 in the kidney of 6-wk old Col1a1(3.6)-Cre;Pkd1^flox/flox caused massive cyst formation, which served as a positive control. Cy, cyst. Scale bars = 100 μm.

Figure 2.

Dmp1-Cre-mediated somatic loss of Pkd1 leads to osteopenia. A) Effects of Dmp1-Cre-mediated Pkd1^Δflox allele on BMD at 6 wk of age. Similar to Beier Pkd1 heterozygous Pkd1^m1Bei/flox mice, there was ∼9–12% reduction of BMD in both female and male single-excised floxed Dmp1-Cre;Pkd1^flox/+ mice compared with age-matched Pkd1^flox/+ control mice, and an even greater reduction (19–27%) in double-heterozygous Dmp1-Cre;Pkd1^flox/m1Bei (Pkd1^Dmp1-cKO) mice, indicating an additive effect of global mutant and conditional deleted Pkd1 alleles on loss of bone mass. B) Effects of Dmp1-Cre-mediated Pkd1^Δflox allele on bone structure of femurs. Micro-CT analysis of the distal femoral metaphyses and midshaft diaphyses revealed that double-heterozygous Pkd1^Dmp1-cKO mice had greater loss in both trabecular and cortical bone than did single Dmp1-Cre;Pkd1^flox/+ and Pkd1^m1Bei/flox heterozygous mice, consistent with additive effects of global mutant and conditionally deleted Pkd1 alleles on bone structure and a direct role of Pkd1 in bone in osteocytes. C) Effects of Dmp1-Cre-mediated Pkd1^Δflox allele on bone MAR. There was a significant decrease in MAR in Pkd1^m1Bei/flox and Dmp1-Cre;Pkd1^flox/+ mice compared with age-matched control mice and an even greater reduction in Dmp1-Cre;Pkd1^flox/m1Bei mice, indicating an additive effect of global mutant and conditionally deleted Pkd1 alleles to impair osteoblast-mediated bone formation. Data represent means ± sd from 5–6 individual mice. *P < 0.05 vs. control Pkd1^flox/+; ^#P < 0.05 vs. Dmp1-Cre;Pkd1^flox/+ and Pkd1^flox/m1Bei.

Figure 3.

Age-dependent effects of Dmp1-Cre-mediated Pkd1^Δflox allele on bone mass. A) Age-dependent effects of Dmp1-Cre-mediated Pkd1^Δflox allele on femoral BMD. B) Age-dependent effects of Dmp1-Cre-mediated Pkd1^Δflox allele on TV of distal femoral metaphyses. C) Age-dependent effects of Dmp1-Cre-mediated Pkd1^Δflox allele on Ct.Th of femoral midshaft diaphyses. Compared with control mice, there was an age-dependent partial recovery of BMD in Dmp1-Cre;Pkd1^flox/+, Pkd1^flox/m1Bei, and Pkd1^Dmp1-cKO mice, respectively. Micro-CT analysis revealed that the increase in bone mass was caused by a recovery in cortical bone thickness, but BV/TV remained significantly lower in these Pkd1-deficient mice at 16 wk of age. Data represent means ± sd from 5 or 6 individual mice. *P < 0.05 vs. control Pkd1^flox/+; ^#P < 0.05 vs. Dmp1-Cre;Pkd1^flox/+ and Pkd1^flox/m1Bei.

Figure 4.

Effects of Dmp1-Cre-mediated Pkd1 deletion on osteoblastic proliferation and maturation ex vivo. A) Total Pkd1 transcripts by real-time RT-PCR from control and Pkd1^Dmp1-cKO osteoblasts during 21 d of culture. Control osteoblasts exhibited a time-dependent increase of total wild-type Pkd1⁺ allele, while conditional Pkd1^Dmp1-cKO osteoblasts showed a proportionate time-dependent increase of Pkd1^Δflox and Pkd1^m1Bei mutant alleles. B) BrdU incorporation. There was no significant change in BrdU incorporation for 6 h between control and Pkd1^Dmp1-cKO osteoblasts, indicating Dmp1-Cre-mediated Pkd1 deletion did not affect proliferation of primary cultured osteoblasts. C) Alkaline phosphatase (ALP) activity. Primary cultured Pkd1^Dmp1-cKO osteoblasts displayed time-dependent increments in ALP activity during 21 d of culture, but ALP activity was significantly lower at different time points compared with control Pkd1^flox/+ osteoblasts. D) Quantification of mineralization. Alizarin Red-S was extracted with 10% cetylpyridinium chloride and quantified as described in Materials and Methods. Primary cultured Pkd1^Dmp1-cKO osteoblasts had time-dependent increments in Alizarin Red-S accumulation during 21 d of culture, but accumulation was significantly lower at different time points compared with control Pkd1^flox/+ osteoblasts. E–H) Gene expression profiles by real-time RT-PCR. Primary cultured Pkd1^Dmp1-cKO osteoblasts in osteogenic differentiation media showed time-dependent increments in osteogenesis but significantly lower at different time points compared to control osteoblasts, evidenced by a significant reduction in osteoblastic markers, including Runx2 (E), Osteocalcin (F), and Dmp1 (G). In contrast, a marked increase of adipocyte markers, such as aP2 (H) at different time points was observed from the Pkd1^Dmp1-cKO osteoblasts under the same differentiation medium when compared with control osteoblasts. Data are expressed as means ± sd from 3 independent triplicate experiments. *P < 0.05 vs. control Pkd1^flox/+.

Figure 5.

Effects of Pkd1 deletion and mutation on baseline and flow-induced [Ca²⁺]_i response in osteoblasts. A, B) Gene dose-dependent reduction of basal [Ca²⁺]_i level was observed in cultured heterozygous Pkd1^null/+ and homozygous Pkd1^null/null cells (n=32; A), as well as heterozygous Pkd1^m1Bei/+ and homozygous Pkd1^m1Bei/m1Bei cells (n=32; B) compared with wild-type Pkd1^+/+ cells (n=32). C, D) Flow-induced [Ca²⁺]_i responses are also impaired in a gene dose-dependent fashion in cultured heterozygous Pkd1^null/+ and homozygous Pkd1^null/null cells (n=10; C) as well as heterozygous Pkd1^m1Bei/+ and homozygous Pkd1^m1Bei/m1Bei cells (n=10; D) compared with wild-type Pkd1^+/+ (n=10) cells. Immortalized osteoblasts from newborn wild-type Pkd1^+/+, heterozygous Pkd1^null/+ and Pkd1^m1Bei/+, and homozygous Pkd1^null/null and Pkd1^m1Bei/m1Bei mice were cultured on type I rat tail collagen-coated 40-mm diameter glass slides at 80–90% confluency. Cells were loaded with 3 μM Fura-2-AM, and the slide was then placed in an FCS2 parallel plate flow chamber. Real-time record of fluorescence intensity (F340/F380 ratio) was performed in the cells when exposed to 6.24 dyn/cm² pulsatile laminar fluid flow. The 340/380 ratios were converted to concentration using standard calibration curve. Data represent means ± sd from individual cells.

Figure 6.

Conditional deletion and mutation of Pkd1 in osteocytes impairs anabolic response to mechanical loading. A) Representative images of midshaft ulnar cross sections from no-load and loaded ulnae of male control Pkd1^flox/+ mice, single Pkd1^flox/m1Bei heterozygous mice, single Dmp1-Cre;Pkd1^flox/+ heterozygous mice, and conditional Pkd1^Dmp1-cKO null mice after loading. There was a robust bone formation response on the medial (inset) and lateral surfaces of loaded control Pkd1^flox/+ ulna. Intermediate bone formation response was observed in the loaded ulnae of Pkd1^flox/m1Bei and Dmp1-Cre; Pkd1^flox/+ mice, but almost no response can be observed in the loaded Pkd1^Dmp1-cKO ulnae. B) MAR on periosteal surface of the midshaft ulna in response to applied mechanical strain in 4 genotypes. Pkd1 gene dose-dependent impairment of MAR was observed in response to −3.0-N loading strains. C) Real-time RT-PCR analysis of total Pkd1 transcripts in no-load ulnae of control and Pkd1-deficient mice at 16 wk of age. Expression of total Pkd1 transcripts was performed using Pkd1-allele-specific primers, as described in Materials and Methods. Similar to the observation in 6-wk-old mice, reduction in total functional Pkd1 transcripts was proportionate to the gene dose in single Pkd1 heterozygous and conditional Pkd1-null mice. D) Expression of mechanical load responsive genes in control and Pkd1^Dmp1-cKO mice 4 h after loading. Real-time RT-PCR analysis from both male genotypes shows that mRNA levels of Runx2-II, Cox-2, c-Jun, and Wnt-related genes (such as Wnt10b, FzD2, and Axin2) were significantly increased in the loaded ulnae from control Pkd1^flox/+ mice, but there was much less change in loaded ulnae from Pkd1^Dmp1-cKO mice. E) Strain gauge measurement. Linear relationship between peak compressive force (N) and peak tension microstrain (με) at the lateral ulna midshaft during cyclic axial compression loading ex vivo was observed from Pkd1^flox/+ and Pkd1^Dmp1-cKO mice. This relationship was significantly different between Pkd1^flox/+ and Pkd1^Dmp1-cKO ulnae using 2-way ANOVA analysis (P<0.0001). Ulnae were loaded in vivo using a peak compressive force of −3.0 N; corresponding microstrain was 1.5-fold greater in Pkd1^Dmp1-cKO ulnae than in control Pkd1^flox/+ ulnae. Data represent means ± sd from 5 or 6 individual mice. *P < 0.05 vs. control Pkd1^flox/+.