The nuclear factor of activated T cells (NFAT) transcription factor NFATp (NFATc2) is a repressor of chondrogenesis

Abstract

Nuclear factor of activated T cells (NFAT) transcription factors regulate gene expression in lymphocytes and control cardiac valve formation. Here, we report that NFATp regulates chondrogenesis in the adult animal. In mice lacking NFATp, resident cells in the extraarticular connective tissues spontaneously differentiate to cartilage. These cartilage cells progressively differentiate and the tissue undergoes endochondral ossification, recapitulating the development of endochondral bone. Proliferation of already existing articular cartilage cells also occurs in some older animals. At both sites, neoplastic changes in the cartilage cells occur. Consistent with these data, NFATp expression is regulated in mesenchymal stem cells induced to differentiate along a chondrogenic pathway. Lack of NFATp in articular cartilage cells results in increased expression of cartilage markers, whereas overexpression of NFATp in cartilage cell lines extinguishes the cartilage phenotype. Thus, NFATp is a repressor of cartilage cell growth and differentiation and also has the properties of a tumor suppressor.

Figure 1

Cartilage masses and joint destruction in NFATp^−/− mice. X ray of a severely affected 20-mo-old NFATp^−/− female (left) and a +/+ female littermate (right). Multiple calcified masses in the shoulder, knee, hip, and ankle joints and joint destruction are present. X-OMAT Diagnostic film (Eastman Kodak, Inc.) was used, and the exposure time was set at 50 s, 20 kV.

Figure 3

Characteristics of cartilage cells derived from NFATp^−/− cartilage. (a) RT-PCR of RNA prepared from wt and NFATp^−/− cartilage cells showing presence of wt and mutant NFATp transcripts as well as transcripts for NFATc, NFAT4, and NFAT3. (b) Phase–contrast micrograph of NFATp^−/− cartilage cells. (c) Immunohistochemical analysis of the same field stained with an antibody to type II collagen. (d) Phase–contrast micrograph of NFATp^−/− cartilage cells. (e) The same field stained with an antibody to COMP.

Figure 2

Cartilage cell proliferation in articular cartilage and in extraarticular connective tissue in NFATp^−/− mice. (A and B) Low power (original magnification: ×20) of the femoral heads and acetabula of 12-mo-old control +/− (A) and NFATp^−/− mutant (B) mice, showing thickening of articular cartilage and sites of extraarticular cartilage cell proliferation and joint destruction in the mutant animal. (C) High power (original magnification: ×200) of B, showing invasion of the acetabulum by proliferating cartilage cells (black arrows) and obliteration of the joint space (white arrows) with loss of safranin-O staining (*). (D) Femoral head and extraarticular connective tissues of 3-mo-old NFATp^−/− mouse (original magnification: ×100), clearly demonstrating the physical separation of the sites of articular and extraarticular cartilage proliferation. (E) Extraarticular connective tissues of 3-mo-old NFATp^−/− mouse demonstrating the differentiation of resident cells into ordered columnar cartilage (arrows). (F) High power (original magnification: ×200) of E. (G) Extraarticular connective tissues of 6-mo-old NFATp^−/− mouse showing calcification of cartilage and the beginnings of endochondral ossification in the extraarticular soft tissue (original magnification: ×200). ac, articular cartilage; fh, femoral head; a, acetabulum; ea, extraarticular; ob, osteoblasts; oc, osteocytes; cc, calcified cartilage; b, bone.

Figure 4

Expression of NFATp is regulated during chondrogenesis. Northern blot analysis of human MSCs undifferentiated or differentiated under chondrogenic (top) or osteogenic (bottom) conditions. Expression of NFATp and actin was determined.

Figure 5

Absence of NFATp enhances, whereas overexpression of NFATp represses the cartilage phenotype. (A) RT-PCR analysis of mature cartilage gene expression in primary wt and NFATp^−/− articular chondrocytes using types II and X collagen and HPRT primers. (B) Western blot analysis of S12 and EA control (Rep) and NFATp transfectants with anti-NFATp antibody. (C) RT-PCR analysis of mature cartilage gene expression of S12 and EA control and NFATp transfectants using type II collagen, type X collagen, CDMP-1, and actin primers (references 36–38).

Figure 5

Absence of NFATp enhances, whereas overexpression of NFATp represses the cartilage phenotype. (A) RT-PCR analysis of mature cartilage gene expression in primary wt and NFATp^−/− articular chondrocytes using types II and X collagen and HPRT primers. (B) Western blot analysis of S12 and EA control (Rep) and NFATp transfectants with anti-NFATp antibody. (C) RT-PCR analysis of mature cartilage gene expression of S12 and EA control and NFATp transfectants using type II collagen, type X collagen, CDMP-1, and actin primers (references 36–38).

Figure 5

Absence of NFATp enhances, whereas overexpression of NFATp represses the cartilage phenotype. (A) RT-PCR analysis of mature cartilage gene expression in primary wt and NFATp^−/− articular chondrocytes using types II and X collagen and HPRT primers. (B) Western blot analysis of S12 and EA control (Rep) and NFATp transfectants with anti-NFATp antibody. (C) RT-PCR analysis of mature cartilage gene expression of S12 and EA control and NFATp transfectants using type II collagen, type X collagen, CDMP-1, and actin primers (references 36–38).

Figure 6

Transformation of and invasion by NFATp^−/− cartilage cells. (A) Extraarticular connective tissues of 6-mo-old NFATp^−/− mouse showing invasion and differentiation of cartilage cells into muscle. Primitive transformed proliferating cells show early differentiation into chondroblasts (arrows); original magnification: ×100. (B) High power (original magnification: ×200) of A, showing columns of primitive transformed cells differentiating into prechondroblasts. (C) High power (original magnification: ×200) of 1-yr-old NFATp^−/− mouse showing cartilage cells invading muscle (arrows). (D) High power (original magnification: ×400) of articular cartilage of 1-yr-old NFATp^−/− mouse showing abnormal-looking cartilage cells with multiple nuclei (arrows). Note the normal appearance of the layer of calcified cartilage. (E) High power (original magnification: ×400) of extraarticular cartilage cells from 1-yr-old NFATp^−/− mouse showing multiple cells in clusters (arrows). (F) Same as E, showing multiple nucleoli (arrows); original magnification: ×400. m, muscle; c, cartilage cells; cc, calcified cartilage; ob, osteoblasts; b, bone.

Figure 7

NFATp^−/− cartilage cells show loss of contact-induced growth inhibition. Varying numbers of wt or NFATp^−/− cartilage cells were plated, and cell division was monitored. Cell division continued even in cells that had reached confluency.