Efficacy of intermittent PTH in WT vs. Ts65Dn mice.

<p>Male mice (N = 6–8 per group) were given daily injections of vehicle or 30 or 80 ug/kg PTH(1–34) for 4 weeks. (<b>A</b>) BMD was measured at the beginning and end of the experiment. (open squares, WT; closed circles, Ts65Dn). Dotted lines show BMD of vehicle treated animals, dashed lines, 30 ug/kg PTH; solid lines, 80 ug/kg PTH (<b>B</b>) % bone volume/tissue volume (BV/TV), trabecular number (Tb.N.) and trabecular thickness (Tb.Th.) were determined in the tibia (open bars WT; solid bars Ts65Dn). Representative superior view of a transverse micro-CT images of the trabecular bone from the proximal tibia of representative animals in each group are shown. (<b>C</b>) Micro CT measurements of the effects of PTH treatment on cortical thickness, periosteal perimeter and endocortical perimeter in the distal tibia were performed (open bars WT; solid bars Ts65Dn). *, p<0.05 vs. respective vehicle control; #, p<0.05 vs. WT vehicle.</p

Histomorphometric measurement of bone formation and bone resorption following intermittent PTH in WT and Ts65Dn mice.

<p>Male mice (N = 6–8 per group) were given daily injections of vehicle or 30 or 80 ug/kg PTH(1–34) for 4 weeks. (<b>A</b>) Mineral apposition rate (MAR) (um³/um²/day), (<b>B</b>) Bone formation rate/bone surface (BFR/BS) (um³/um²/day), (<b>C</b>) Number of osteoblasts/bone perimeter (N.Ob./B.Pm), (<b>D</b>) Number of osteoclasts/bone perimeter (N.Oc./B.Pm.) were measured in WT and Ts65Dn mice vehicle or PTH treated (30 or 80 ug/kg/day) (open bars, WT; closed bars, Ts65Dn). *, p<0.05 vs. respective vehicle control; #, p<0.05 vs. WT vehicle.</p

<i>Sc65KO</i> mouse generation and confirmation of bone loss phenotype.

<p>a) Strategy for the creation of the <i>Sc65-null</i> allele. The schematic diagram shows the <i>Sc65</i> wild-type, targeted, floxed and excised allele. <i>Sc65</i> coding regions are in light blue while non-coding regions are in dark blue. Also note the proximity to the <i>Fkbp10</i> gene which is transcribed in the opposite orientation. b) PCR genotyping of <i>Sc65KO</i> mice (upper panel) and Western blot confirmation of SC65 protein (arrow) loss in multiple <i>Sc65KO</i> tissues from 3 day-old mice compared to WT controls (lower panel—Cal = calvaria, Kid = Kidney). c) Immunohistochemistry detection of SC65 in adult femur section from a WT mouse showing specific intracellular staining in bone forming cells (osteoblasts) aligned on the surface of a bone trabecula. <i>Sc65</i> expression is lost in a similar section from a <i>Sc65KO</i> mouse. Scale bars = 100μM (10x) or 20μM (63x). d) MicroCT analysis of long bones from 6 month-old WT and <i>Sc65KO</i> male mice (n = 9). Both femurs and tibias from <i>Sc65KO</i> mice exhibited decreased trabecular bone volume/tissue volume (BV/TV), connectivity density (Conn.D) and cortical thickness (Ct.Th) compared to WT controls (*p<0.05).</p

SC65 directly interacts with prolyl 3-hydroxylase 3 (P3H3).

<p>a) Lysates of 714 mouse embryonic fibroblasts stably expressing SC65-Flag or EV control were used for IP experiments utilizing a Flag antibody (upper panel) or a P3H3 antibody (lower panel). 10% of total inputs and immuno-precipitates were separated on a 10% SDS-PAGE gel, blotted and probed with antibodies against FLAG and P3H3. The reciprocal interaction of SC65-Flag with P3H3 is confirmed in both experiments. b) Western blot of primary calvarial osteoblast and skin fibroblast lysates from WT and <i>Sc65KO</i> 3 day-old mice (N = 2) showing significantly decreased levels of P3H3 protein in <i>Sc65KO</i> samples. Densitometric quantification of P3H3 protein normalized to β-actin from the western blot shown above (#p<0.01; *p<0.05; error bars represent SD). All experiments were performed at least 3 times.</p

Loss of Sc65 results in dermal tears, abnormal collagen fibrils and skin fragility.

<p>a) H&E stained sections of WT and <i>Sc65KO</i> skin. Note the decreased density of collagen, the frayed dermis indicated by arrows and the reduced thickness of the muscle layer in the <i>Sc65-null</i> samples. b) Serial skin sections were stained with Sirius red. <i>Sc65-null</i> skin exhibits fewer large collagen fibers (red staining) and greater number of smaller collagen fibers stained in green compared to WT counterparts. c) Electron micrographs of 7 month-old mouse skin biopsy from WT and <i>Sc65KO</i> mice. Collagen fibrils, shown in cross-section, from <i>Sc65-null</i> skin tended to be smaller and have a decreased range of fibril diameter compared to WT fibrils. Loss of Sc65 also resulted in the presence of collagen fibrils with irregular profile and several large “cauliflower-like” fibrils (red arrow) which indicate abnormal fibrillogenesis (scale bar represents 500nm). d) Distribution of collagen fibril diameter in WT and <i>Sc65KO</i> mouse skin as measured from electron microscopy images. Measurements were collected from three different mice/genotype and >200 fibril/mouse. e) Skin EMs from <i>Sc65KO</i> mice also exhibited significantly more empty space among collagen fibrils compared to WT mice indicating a less densely packed collagen (*p = 0.01). Five electron micrograph images of non-overlapping areas were quantified from each mouse. f-h) Skin samples from WT and <i>Sc65KO</i> mice were subjected to a biomechanical skin loading test to measure tensile strength. Skin that lacks SC65 expression ruptured at a significantly lower peak load compared to WT skin indicating significant skin fragility (*p<0.01).</p

Increased electrophoretic mobility and altered cross-linking of type I collagen from <i>Sc65-null</i> skin.

<p>a) SDS-6%PAGE of type I collagen extracted from skin and decalcified bone of <i>Sc65KO</i> and WT mice shows increased mobility of α-chains and reduced ratio of cross-linked β to γ components in the <i>Sc65KO</i> skin extracts. An acetic acid extract from skin of the original Sc65-null mouse¹⁹ (1 mo.) created by gene-trap insertion is compared with that from the new <i>Sc65KO</i> (6 mo.) and their respective WT controls. Total heat denatured extracts of skin and bone collagens from new <i>Sc65KO</i> mice are shown on the right for comparison. Bone collagen from <i>Sc65KO</i> mice does not show the differences from WT in β/γ intensities evident for skin collagen. The strong (lower) γ band in SC65 skin extracts was identified as γ _112. Both original and new <i>Sc65KO</i> mice showed the same altered pattern of chain intensities from WT most pronounced in the acetic acid extracts of skin with an apparent increase in γ₁₁₂ at the expense of β_12. b) Densitometric analysis of collagen bands on SDS-PAGE. Densitometry was performed on bands 1–8 (counted from top to bottom) of acetic acid extracts from 1mo and 6mo skin samples of both original and new <i>Sc65KO</i> mice using NIH imageJ software. Values are means ± SD, n = 6; *p<0.01.</p

Osteoclast and osteoblast formation is significantly decreased in 3 month old Ts65Dn Mice.

<p>(<b>A</b>) <i>Ex vivo</i> recruitment into the osteoblast lineage was measured at culture day 10 by staining for alkaline phosphatase (AP) and counting the number of AP+ colony forming units (CFU-F) per well. (<b>B</b>) <i>Ex vivo</i> osteoblast differentiation was assessed at culture day 28 by staining mineralized bone nodules containing differentiated colonies of osteoblasts (CFU-OB) with Alizarin Red and the number of CFU-OB per well enumerated. (<b>C</b>) <i>Ex Vivo</i> osteoclast differentiation was assessed by staining on day 14 for tartrate resistant acid phosphatase (TRAP) and the number of TRAP+-multinucleated cells per well counted. *, p<0.05 vs. WT vehicle control.</p

SC65 directly interacts with lysyl-hydroxylase 1 (LH1).

<p>a) Murine 714 mouse embryonic fibroblasts stably expressing SC65-Flag or an empty vector (EV) control were transiently transfected with a HA-tagged Lh1 and lysed 48 hours after transfection. Immuno-precipitation (IP) experiments were conducted using a Flag antibody (upper panel) or a HA antibody (lower panel). 10% of total inputs and immuno-precipitates were separated on a 10% SDS-PAGE gel, blotted and probed with antibodies against FLAG and HA. The reciprocal interaction of SC65-Flag with LH1-HA is confirmed in both experiments. b) Western blot of primary calvarial osteoblast and skin fibroblast lysates from WT and <i>Sc65KO</i> 3 day-old mice (N = 2) showing significantly decreased levels of LH1 protein in <i>Sc65KO</i> samples. Densitometric quantification of LH1 protein normalized to β-actin from the western blot shown above (*p<0.05; error bars represent SD). All experiments were performed at least 3 times.</p

Schematic representation of a fibrillar collagen molecule in the ER.

<p>The long uninterrupted triple-helical domain is shown here already folded and without N- and C-propeptides. Depicted in different colors are some of the prolyl 3-hydroxylase enzymes (P3Hs), lysyl hydroxylase enzymes (LHs) and cyclophilin B (purple) with some of their known substrate residues. Our previous work identified the prolyl 3-hydroxylation complex that modifies P986; our current work suggests the existence of a new complex, possibly including CYPB, responsible for the hydroxylation of K87 and K930. Evidence indicates that CRTAP and SC65 act as unique orchestrators of essential molecular complexes for collagen post-translational modification. (LH2 is likely to also act within a protein complex but direct interactions have not yet been published).</p

Characterization of a new SC65/LH1/P3H3 complex in the ER.

<p>a) Lysates of 714 mouse embryonic fibroblasts that were transiently transfected with an HA-tagged LH1 expression construct were immuno-precipitated with a HA antibody (upper panel) or a P3H3 antibody (lower panel). 10% of total inputs and immuno-precipitates were separated on a 8% SDS-PAGE gel, blotted and probed with antibodies against HA and P3H3. Negative controls included non-transfected 714 cells incubated with the HA antibody (for non-specific binding of HA antibody, left lanes) and LH1-HA transfected cells incubated with no antibody (for non-specific proteins binding to beads, middle lanes). In both experiments, LH1-HA and P3H3 were found to interact (right lanes). b) Lysates of 714 mouse embryonic fibroblasts stably expressing SC65-Flag or EV control and transiently transfected with a HA-tagged CYPB were used for IP utilizing an HA antibody. 10% of total input and immuno-precipitates were separated on a 12% SDS-PAGE gel, blotted and probed with antibodies against Flag and HA. The blot detecting SC65-Flag following IP with the HA antibody is shown over-exposed. c) Western blot of primary calvarial osteoblast and skin fibroblast lysates from WT and <i>Sc65KO</i> 3 day-old mice (N = 2) showing similar content of CYPB protein in <i>Sc65KO</i> and WT samples. All experiments were performed at least 3 times.</p