Functional analysis of insulin-like growth factor binding protein -4 and -6 in transgenic mice

Insulin-like growth factors (IGF-I and IGF-II) are expressed in
many cell types and tissues and act in endocrine, autocrine or
paracrine manner to regulate cellular proliferation, survival and
differentiation. IGF actions are initiated upon binding to the type
I IGF receptor (IGF-IR) and are modulated through interactions with
a family of six secreted IGF-binding proteins (IGFBP-1 to -6).
Although the six conserved IGFBPs are structurally related, each of
them has specific characteristics and may have specific functions.
Most knowledge about the IGFBPs has been gained from the numerous
in vitro studies, their specific roles in vivo are largely unknown.
Transgenic mice overexpressing a particular IGFBP allow us to
investigate the specific functions of the corresponding IGFBP in
vivo. To this end, IGFBP-4- and IGFBP-6-overexpressing models were
established and analyzed in the present study. First, an expression
vector containing the murine H-2Kb promoter and a human beta-globin
splicing cassette was used to construct the transgenes, to obtain
ubiquitous expression of the mouse Igfbp4 and Igfbp6 cDNA. Two
lines of H-2Kb-mcIGFBP-4 and ten lines of H-2Kb-mcIGFBP-6
transgenic mice were generated. The transgene was ubiquitously
expressed at RNA level in both transgenic models, however, at
protein level, transgene expression was only detected in the
spleen, thymus, lung and kidney of both H-2Kb-mcIGFBP-4 transgenic
lines, but in no organ of H-2Kb-mcIGFBP-6 transgenic mice.
Phenotypic analyses of the H-2Kb-mcIGFBP-4 transgenic model
revealed that overexpression of IGFBP-4 had no significant effect
on the postnatal body and organ growth, except that the weight and
volume of thymus in 8- and 12-week-old transgenic mice were
significantly reduced (p < 0.05) compared to the controls.
Histomorphometric analysis demonstrated that the volume of the
thymic cortex was significantly decreased in transgenic mice (p
< 0.05), whereas that of the thymic medulla was not changed. The
fractions of various cell types in the bone marrow, thymus, spleen,
lymph node and peripheral blood were determined by flow cytometry.
No significant difference was found between transgenic and control
groups, suggesting that IGFBP-4 excess in the lymphoid organs did
not affect the development of the lymphatic cells. The
proliferative capacity of the splenocytes of transgenic animals was
significantly reduced after Con A and LPS stimulation (p <
0.05), but not altered after the stimulation by anti-CD3 and
anti-IgM/IL2. This is probably due to transgenic IGFBP-4 expression
restricted in the non-lymphatic cells. However, detailed expression
of the transgene warrants further investigation. In order to
realize IGFBP-6-overexpressing mice, a second construct was
designed, namely CMV-mgIGFBP-6, in which the mouse Igfbp6 genomic
sequence was cloned under the control of the cytomegalovirus (CMV)
promoter. Four independent lines of transgenic mice were generated.
Transgene expression was high in the exocrine pancreas and
relatively low in the lung and liver. The activities of serum
IGFBPs were not different between transgenic mice and controls. In
transgenic mice, high levels of active IGFBP-6 were detected in the
luminal content of the duodenum, but neither in the luminal
contents of other segments of the gastrointestinal tract (GIT), nor
in tissue extracts of all GIT segments. Glucose homeostasis was not
altered by IGFBP-6 expression. Postnatal body and organ growth was
not affected in transgenic mice, except for the absolute and
relative weight and length of duodenum which were significantly
reduced in 4-month-old transgenic mice as compared to controls (p
< 0.05). This reduction was mainly due to a significantly
smaller volume and surface area of the tunica mucosa as determined
by histomorphometric analsis. Our analysis of the first IGFBP-6
transgenic mouse model provides direct evidence for inhibition of
intestinal growth by luminal IGFBP-6 excess. This finding is
important in the context of neonatal intestinal growth of mammals,
considering the fact that milk contains large amount of IGFBPs
which may at least in part arrive intact in the intestine.