Connective tissue growth factor is required for skeletal development and postnatal skeletal homeostasis in male mice

Abstract

Connective tissue growth factor (CTGF), a member of the cysteine-rich 61 (Cyr 61), CTGF, nephroblastoma overexpressed (NOV) (CCN) family of proteins, is synthesized by osteoblasts, and its overexpression inhibits osteoblastogenesis and causes osteopenia. The global inactivation of Ctgf leads to defective endochondral bone formation and perinatal lethality; therefore, the consequences of Ctgf inactivation on the postnatal skeleton are not known. To study the function of CTGF, we generated Ctgf(+/LacZ) heterozygous null mice and tissue-specific null Ctgf mice by mating Ctgf conditional mice, where Ctgf is flanked by lox sequences with mice expressing the Cre recombinase under the control of the paired-related homeobox gene 1 (Prx1) enhancer (Prx1-Cre) or the osteocalcin promoter (Oc-Cre). Ctgf(+/LacZ) heterozygous mice exhibited transient osteopenia at 1 month of age secondary to decreased trabecular number. A similar osteopenic phenotype was observed in 1-month-old Ctgf conditional null male mice generated with Prx1-Cre, suggesting that the decreased trabecular number was secondary to impaired endochondral bone formation. In contrast, when the conditional deletion of Ctgf was achieved by Oc-Cre, an osteopenic phenotype was observed only in 6-month-old male mice. Osteoblast and osteoclast number, bone formation, and eroded surface were not affected in Ctgf heterozygous or conditional null mice. In conclusion, CTGF is necessary for normal skeletal development but to a lesser extent for postnatal skeletal homeostasis.

Figure 1

Engineering of the Ctgf^e2COIN allele and strategy for the conditional inactivation of Ctgf. The upper panel reveals the exon and intron structure of Ctgf (adapted from Ensembl.org). Dark gray boxes indicate coding sequences, whereas white boxes indicate untranslated regions (UTR). The introns are shown as dotted lines. In the Ctgf^e2COIN allele, exon 2 (223 bp) is split by inserting a COIN intron into two new exons of 120 and 103 bp upstream and downstream of the COIN intron, respectively. The COIN intron contains a COIN element that is comprised of lox66_SA-Egpf-polyA_lox71, placed in the antisense orientation with respect to the transcription of the Ctgf. A single FLP recognition target (FRT) site indicates the placement of the HygΔTK drug selection cassette, which was removed by the action of FLP. A normal CTGF mRNA is expressed by the Ctgf^e2COIN allele. The middle panel shows that exposure to Cre recombinase results in the virtually irreversible inversion of the COIN element and conversion of the lox66-lox71 pair to lox71-loxP. A new message is expressed, comprised of exons 1, the new exon 2, and COIN element exon, which encodes for a transmembrane domain-eGFP fusion protein (TMeGFP). In the lower panel, a representative PCR analysis, using primers 1, 2, and 3 (P1, P2, and P3) depicted in the upper and middle panels and described in Supplemental Table 1, is shown. Calvarial DNA from Ctgf conditional null and control mice before and after recombination by Cre expressed under the control of the Prx1 enhancer (left panel) or of the osteocalcin promoter (right panel) is shown. A 710-bp band is detected in the Ctgf^INV allele, and a 650-bp band is detected in the noninverted allele.

Figure 2

Weight, femoral length, BMD, and Ctgf mRNA expression in male (upper panels) and female (lower panels) Ctgf^+/LacZ heterozygous null mice (•) and wild-type littermate controls (○). The weight in grams (A), femoral length in millimeters (B), total BMD in grams per square centimeter (C), and Ctgf mRNA levels in total calvarial extracts (D), expressed as Ctgf copy number corrected for Rpl38 and normalized to 100 at 1, 4, and 6 months of age, are shown. Values are means ± sem (n = 5–17) except for mRNA levels, which are expressed as percentage of control for each independent age (n = 3–4). * Significantly different from control mice, P < 0.05.

Figure 3

Representative histological sections and calcein/demeclocycline labeling of bone femoral sections from 1-month-old Ctgf^+/LacZ heterozygous mice (A), 1-month-old Prx1-Cre/+;Ctgf^INV/INV conditional null mice and littermate Ctgf^{e2COIN/e2COIN} controls (B), and 6-month-old Oc-Cre/+;Ctgf^INV/LacZ conditional null mice and Oc-Cre/+;Ctgf^INV/+ littermate controls (C). Sections from male mice were stained with von Kossa without counterstain (final magnification, ×40) or unstained and examined under fluorescence microscopy (final magnification, ×400). WT, Wild type.

Figure 4

Weight, femoral length, BMD, and Ctgf expression in male and female Prx1-Cre/+;Ctgf^INV/INV conditional null mice (black bars) and littermate Ctgf^{e2COIN/e2COIN} controls (white bars). The weight in grams (A), femoral length in millimeters (B), total BMD in grams per square centimeter (C), and Ctgf mRNA levels in total calvarial extracts (D), expressed as Ctgf copy number corrected for Rpl38 and normalized to 100, are shown. Values are means ± sem (n = 3–6), except for RNA levels, which are expressed as percentage of control (n = 2–6).

Figure 5

Weight, femoral length, BMD, and Ctgf expression in male (upper panels) and female (lower panels) Oc-Cre/+;Ctgf^INV/LacZ conditional null mice (•) and Oc-Cre/+;Ctgf^INV/+ littermate controls (○). The weight in grams (A), femoral length in millimeters (B), total BMD in grams per square centimeter (C), and Ctgf mRNA levels in total calvarial extracts (D), expressed as Ctgf copy number corrected for Rpl38 and normalized to 100 at 1, 4, and 6 months of age, are shown. Values are means ± sem (n = 4–13) except for mRNA levels, which are expressed as percentage of control for each independent age (n = 2–6). *, Significantly different from controls by unpaired t test, P < 0.05.