Modest effects of ACTH on osteoclast precursors. (A) Human CD14 osteoclast precursors express significant amounts of mRNA for the ACTH receptor MC2R, although much less than that expressed on human osteoblasts (see Fig. 3E). However, during RANKL-induced differentiation, osteoclasts lose MC2R and would thus be expected to respond poorly to ACTH. (B) Very low levels of MC2R expression measured by quantitative PCR (qPCR) in mature human osteoclasts, with or without ACTH (10⁻⁸ M). (C) Human CD14 cells show a modest increase in TNFα mRNA with ACTH, suggesting a small effect on osteoclastogenesis. (D) In keeping with the response of human cells, a 5-day treatment of murine bone marrow cells with ACTH (concentrations as shown) caused a modest but clear increase in numbers of tartrate-resistant acid phosphatase- (TRAP-) positive osteoclasts, but without a clear concentration-dependence. (E) In contrast, RAW264.7 cells displayed a clear concentration-dependent increase in osteoclast formation. This transformed cell line retains large numbers of division-competent uncommitted precursor cells, even in the presence of RANKL. (F) qPCR for early osteoclast differentiation genes in response to RANK-L or RANK-L plus ACTH, namely tartrate-resistant acid phosphatase (TRAP), cathepsin K (Cath K), NFATc1, metalloproteinase (MMP) 9 and 13, and the inhibitors (IκBα and IκBβ) and subunits (p65, p 105, and c-Rel) of NF-κB. (G) qPCR for late differentiation markers, such as integrin β₃ and the calcitonin receptor (CTR), and control proteins, such as NFATc2 and NFATc4. Statistics: Student's t test, *P < 0.05, **P < 0.01, qPCR in triplicate; TRAP⁺ osteoclasts (n = 8 wells per treatment).

ACTH protects the rabbit femoral head from glucocorticoid-induced focal osteonecrosis. Rabbits (approximately 4.5 kg) were treated with 10 mg/kg depot methylprednisolone acetate (MPA), and half of the animals also received 0.2 μg/kg ACTH (1–24) (cosyntropin) daily. (A) Dual energy x-ray absorptiometry (DXA) scans of femoral heads after 24 days of MPA with or without ACTH (a and b) showed regions of nonuniform density that were slightly more apparent in MPA-only group. Microcomputed tomography (c and d) revealed focal damaged trabeculae, indicating rapid resorption in the MPA-treated group (arrow). (B) Femoral head bone mineral density measurements showed only a minor overall difference; however, there was a 30% increase in the subarticular bone-forming surfaces on tetracycline labeling in the MPA plus ACTH group (n = 4, mean ± SD, not significant). (C) Although tetracycline labels bone forming surfaces (strong linear labeling), it is taken up passively by necrotic bone as shown (6). The region from the articular cartilage, 2- to 3-mm deep, showed only surface tetracycline labeling and was protected from necrosis (a and b). In contrast, in the centers of the femoral heads, all animals had focal necrotic bone with diffuse tetracycline labeling of osteons (c and d), extending in many cases entirely through trabeculae. In the group without ACTH (c), however, there were prominent consolidated areas of necrosis, in which marrow adipocytes were also necrotic, causing lipolysis that also bound calcium and, thus, labeled with tetracycline. The results were in keeping with the discrete lesions noted on μCT in the MPA-only group (Ac). With ACTH, areas of necrosis were smaller and generally not consolidated with less prominent labeling of necrotic adipocytes. (D) As noted, tetracycline labels bone formation at the mineralizing surface, but necrotic bone loses its isolation from extracellular fluid, causing diffuse labeling of the necrotic bone (a). There was a highly significant approximately 50% reduction in the necrotic surface area in femora from rabbits treated with MPA plus ACTH compared with those given MPA alone (c); these data demonstrate a clear protection of the focal osteonecrosis by ACTH. Statistics: Student's t test; *P < 0.05; n = 4; mean ± SD). Effect of ACTH on VEGF mRNA expression measured by quantitative PCR in MPA-treated rabbits (d). Statistics: Student's t test; *P < 0.05; in triplicate; mean ± SD).

ACTH promotes osteoblast differentiation and protects against glucocorticoid-induced apoptosis. (A) ACTH (10⁻⁸ M) increases the expression of genes associated with osteoblast maturation and activity, including alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin, the key transcription factor for osteoblast differentiation Runx2, as well as the osteoclastogenic cytokine RANK-L and its decoy receptor, osteoprotegerin (OPG), after 28 but not 10 days of culture in medium containing 100 μM ascorbate and 10 mM glycerol phosphate (“differentiation medium”). Statistics: Student's t test, *P < 0.05, **P < 0.01; mean ± SEM, in triplicate. (B) Human mesenchymal stem cells in differentiation medium for 28 days showed typical morphological changes with ACTH and dexamethasone (Dex), namely cuboidal cell transformation and rapid mineralization (17) (fields: 440 μm²), measured as the cell's aspect ratio (long/short axis). Statistics: Student's t test, mean ± SD, n = 10, *P < 0.05, ^P = 0.012 (C). (D) Quantitative PCR on similar 28-day cultures showed effects on osteoblast maturation genes consistent with those in murine bone stromal cells (A), notably increases in type 1 COLLAGEN (Col1), RUNX2, and VEGF mRNA. However, the expression of the ACTH receptor MC2R was attenuated (i). The overall response of the transformed osteoblast-like cell line MG63 to ACTH was qualitatively similar to that of nontransformed cells (ii), although the response magnitudes were smaller and, in some cases, did not reach significance (as shown). MG63 cells display features of early osteoblastic maturation, but typically do not mineralize. Statistics for C and D: Student's t test, *P < 0.05; mean ± SD. (E and F) Human mesenchymal stem cell cultures in differentiation medium were treated with ACTH (10⁻⁸ M) and/or dexamethasone (Dex, 10⁻⁷ M) (as shown) for 28 days, and apoptosis was measured by labeling with fluorescent annexin V. Only small numbers of apoptotic cells were seen per field (field: 220 μm²). However, when ACTH was withdrawn (w/d) for 36 h, the numbers of annexin V-positive cells increased dramatically, suggesting that ACTH protects against glucocorticoid-induced apoptosis.

ACTH stimulates VEGF production from osteoblasts. (A) Immune precipitation of VEGF from nontransformed human osteoblast culture supernatants, showing that VEGF production is responsive to ACTH (10⁻⁸ M) and, to a lesser extent, to dexamethasone (Dex, 10⁻⁷ M). Media were changed for the last 36 h in the 4-week culture, during which time ACTH was withdrawn (w/d) and VEGF production receded to unstimulated levels. The major ACTH fragment is a well-recognized 28-kDa fragment (20); recombinant VEGF is shown as the approximately 18-kDa fragment. (B) Mineralizing human osteoblasts were rendered quiescent overnight in serum-free medium and treated for the times indicated (hours) with 10⁻⁸ M ACTH. VEGF expression, measured by quantitative PCR, was induced strongly within 1 h, but dropped after 3–4 h. The level of expression was similar to that seen in long-term ACTH-stimulated cultures (see E), although the rapid induction by ACTH of VEGF is seen more clearly in quiescent cells. Basal VEGF production (as in A) may thus be stimulated by serum cytokines or by low levels of ACTH in serum used in osteoblast cultures. (C) Mineralizing nodules formed in human mesenchymal stem cell (MSC) cultures incubated in media permitting osteoblast differentiation for 28 days also display CD31-expressing endothelial cells in capillary patterns similar to the rete of capillaries surrounding bone trabeculae in vivo. Red channel: CD31 label (arrows point to capillaries); overlay of red with the strong collagen signal (green) of the nodule (representative images; fields: 220 μm²). (D) Western blots showing very high VEGF expression in mixed human monocyte (MF)/osteoblast (HOB) cultures for 10 days treated with parathyroid hormone (PTH) and 1.25-(OH)₂ vitamin D (PTH+D), dexamethasone (10⁻⁷ M, Dex) and/or ACTH (10⁻⁸ M). This VEGF production is abrogated by Dex. The importance of this finding is that VEGF production due to monocyte-osteoblast interaction in bone is profoundly down-regulated by glucocorticoids, heightening the potential sensitivity of VEGF production to reduced circulating ACTH. (E) Human monocytes in culture bear low levels of the ACTH receptor MC2R and produce small amounts of VEGF, confirming that the major production is from MC2R-bearing osteoblastic cells. (F) Western blot showing the effect of ACTH (10⁻⁷ M) on VEGF-A production in MC3T3.E1 osteoblastic cells and its knock down by an shRNA. (G) Quantitative PCR showing a lack of effect of ACTH (10⁻⁸ M) on alkaline phosphatase (Alk Phos) or bone sialoprotein (BSP) expression in VEGF-A shRNA-treated cells compared with empty vector-treated cells (EV). Statistics: Student's t test, mean ± SEM, triplicate, *P < 0.01.