Mutation of Prkar1a causes osteoblast neoplasia driven by dysregulation of protein kinase A

Abstract

Carney complex (CNC) is an autosomal dominant neoplasia syndrome caused by inactivating mutations in PRKAR1A, the gene encoding the type 1A regulatory subunit of protein kinase A (PKA). This genetic defect induces skin pigmentation, endocrine tumors, myxomas, and schwannomas. Some patients with the complex also develop myxoid bone tumors termed osteochondromyxomas. To study the link between the PRKAR1A mutations and tumor formation, we generated a mouse model of this condition. Prkar1a(+/-) mice develop bone tumors with high frequency, although these lesions have not yet been characterized, either from human patients or from mice. Bone tumors from Prkar1a(+/-) mice were heterogeneous, including elements of myxomatous, cartilaginous, and bony differentiation that effaced the normal bone architecture. Immunohistochemical analysis identified an osteoblastic origin for the abnormal cells associated with islands of bone. To better understand these cells at the biochemical level, we isolated primary cultures of tumoral bone and compared them with cultures of bone from wild-type animals. The tumor cells exhibited the expected decrease in Prkar1a protein and exhibited increased PKA activity. At the phenotypic level, we observed that tumor cells behaved as incompletely differentiated osteoblasts and were able to form tumors in immunocompromised mice. Examination of gene expression revealed down-regulation of markers of bone differentiation and increased expression of locally acting growth factors, including members of the Wnt signaling pathway. Tumor cells exhibited enhanced growth in response to PKA-stimulating agents, suggesting that tumorigenesis in osteoblast precursor cells is driven by effects directly mediated by the dysregulation of PKA.

Figure 1

Gross and Immunohistochemical Characterization of Bone Tumors in Prkar1a^+/− Mice A, X-ray of a mouse with early tail tumors showing effacement of the normal vertebral bone; B, tail of WT (left) and Prkar1a^+/− (right) mice stained for bone and cartilage with Alizarin red (bone) and Alcian blue (cartilage); C–E, immunohistochemical analysis of bone tumors for Runx2 (C), a marker of early osteoblast differentiation; osteocalcin (D), a marker of late osteoblast maturation; and V-ATPase (E), a marker for osteoclasts (black arrows); F, quantitation of osteoclast numbers per HPF in the bone tumors. The graph shows the average counts from 15 HPF from two WT and two tumor tails. In C and D, tumor cells are shown with black arrows, whereas normal staining osteoblasts are shown with black arrowheads.

Figure 2

Analysis of PKA in Primary Osteoblasts Proteins were prepared from primary cultures of tumor or WT vertebral osteoblasts, as were single samples from primary cultures from heterozygous Prkar1a^+/− mouse femurs (Het Fmr) or WT femurs (WT Fmr). A, Samples were Western blotted for PKA subunits as indicated at right. Actin was used as a loading control. B, Quantitation of the data for the four tumor and three WT vertebral samples. **, P < 0.01. This experiment was repeated three times, and a representative blot is shown. Note that all subunits tested demonstrated statistically significant changes with the exception of Prkar2a. C, PKA activity from WT or tumor cells was measured either in the absence of exogenous cAMP (free PKA activity) or in the presence of 5 μm cAMP (total PKA). P values for the comparisons are shown. In both cases, the difference between free and total levels of PKA activity were not significant.

Figure 3

Morphological and Functional Analysis of Primary Tumor Cells from Prkar1a^+/− Mouse Bone Tumors A–C, Staining for alkaline phosphatase activity of primary cultures of cells isolated from control (WT) primary osteoblasts (A), primary tumor cells (B), and primary mouse embryonic fibroblasts (C); D and E, von Kossa staining of mineralization assay for WT osteoblasts (D) and tumor cells (E) (note that the mineralized nodules, black, fail to condense in the tumor cells); F and G, immunofluorescence for osteocalcin in WT osteoblasts (F) and tumor cells (G). Each of these assays was performed three to five times, with the exception of the immunofluorescence study, which was performed twice. Representative data from each assay are shown.

Figure 4

Analysis of Chondrocyte Markers in Primary Tumor Cells WT or Prkar1a^+/− tumor osteoblasts were isolated from mouse tails and grown in chondrocyte (CHN) or osteoblast (OBL) medium and analyzed by semiquantitative RT-PCR for the mRNAs shown at left. Gapdh was used as a normalization control. Note the marked increase in the chondrocyte markers aggrecan and collagen2 in the tumor osteoblasts. Shown are representative data from replicate experiments.

Figure 5

Injection of Tumor Cells Can Recapitulate Tumor Formation in Immunocompromised Mice A, WT osteoblasts injected sc over right hip (blue arrow) of NOD-SCID mice fail to produce tumors, whereas tumor osteoblasts injected over the left hip (red arrow) produce calcifying tumors; B and C hematoxylin- and eosin-stained sections of the tumors are shown at ×40 (B) and ×400 (C) magnification and demonstrate marked similarity to the initial tumor (e.g. Fig. 1); D–F, eutopic injection of tumor cells into the tail can also produce tumors in mice, shown by x-ray (D, red arrow), or by hematoxylin and eosin at ×40 (E) or ×400 (F); G, normal mouse tail for comparison. D, Intervertebral disk; T, tumor; V, vertebrae.

Figure 6

Analysis of Selected Proteins by Western Blot Proteins were prepared from the same samples as in Fig. 2 and Table 1 and Western blotted for the proteins indicated. Quantitation is shown below as in Fig. 2. Note that all tested proteins showed statistically significant changes, matching the results of mRNA expression analysis. *, P < 0.05; **, P < 0.01. This experiment was repeated five times, and a representative blot is shown.

Figure 7

Analysis of Growth Characteristics of Control and Tumor Osteoblasts A, WT or tumor cells were plated in triplicate in 12-well dishes and allowed to grow in serum-free medium containing no additives (SF), FSK, insulin (Ins), or both (Fsk+Ins) or in complete medium containing 10% fetal calf serum (CM). After 5 d, cells were quantitated. B, Cells were grown in serum-free medium for 3 d with the addition of FSK, PTHrP, or PKI as indicated or in complete medium (CM). Doses of drugs are described in Materials and Methods. **, P < 0.001 for comparison of WT and tumor cells receiving the same treatment.